Serological diagnosis of protostrongylidae infections and identification of unique antigens

ABSTRACT

Novel Protostrongylidea antigens and early and accurate diagnostic methods for Protostrongylidae infection are disclosed. Novel  P. tenuis -specific antigens and methods of discriminating between  P. tenuis  infection and infection with other closely-related members of the Protostrongylidae family are provided. Novel  E. cervi -specific antigens and methods of discriminating between  E. cervi  infection and infection with other closely-related members of the Protostrongylidae family are provided.

PRIORITY CLAIM

[0001] This invention claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application 60/094,064, filed Jul. 24, 1998 and U.S.Provisional Application 60/094,117 filed Jul. 24, 1998, both of whichdisclosures are incorporated by reference in their entireties herein.

BACKGROUND

[0002] Infection with meningeal and tissue worms (Nematoda:Protostrongylidae) can result in serious neurological disease or deathin certain ungulate hosts. The Protostrongylidae family of nematodesincludes a variety of infectious species, for example,Parelaphostrongylus tenuis (meningeal worm), Parelaphostrongylusandersoni, Parelaphostrongylus odocoilei, Elaphostrongylus rangiferi andElaphostrongylus cervi (tissue worm) (see, e.g., Platt (1984) for adiscussion of various protostrongylidae). The nematodeParelaphostrongylus tenuis (P. tenuis) is widespread throughout wildwhite-tailed deer hosts (WTD; Odocoileus virginianus) in the easternhalf of North America. (Anderson et al. (1992), Anderson and Prestwood(1981), Anderson and Streveli (1967)). Although symptoms may be mild ininfected WTD, P. tenuis infection can result in fatal neurologicaldisease in various cervids and camelids as well as in other wild anddomesticated ruminants (Anderson and Prestwood, (1981)).

[0003] Development of a reliable and sensitive method for diagnosinginfection with various meningeal worms has proven difficult. Currently,diagnosis is performed using the Baermann technique or a modifiedBaermann technique (see, e.g., Gajadhar et al. (1994) Can. Vet. J.35:433-437). In brief, this method involves detecting the presence ofdorsal-spined, first-stage larvae (L1) in the feces of infected animals.However, there are several limitations to this technique. In particular,the method does not routinely allow detection of low level infection,although these animals may exhibit clinical signs and even die (Dew etal., 1992; Samuel et al., 1992). The reasons that infected animals mayfail to shed detectable numbers of larvae has not been entirelyelucidated. The parasite load may be low or larvae shed intermittently(Welch et al., 1991). Alternatively, animals infected with only oneworm, or worms of the same gender, will not shed larvae.

[0004] The Baermann technique also fails to provide early detection ofinfection. Because the pre-patent period is long, between 82-137 days(Rickard et al., 1994), infection may not be diagnosed for many monthsand repeated testing is often required. Yet another drawback of theBaermann method is that is does not distinguish between various speciesof the protostrongylidae family. Many of the dorsal-spined larvaedetected in feces are morphologically indistinguishable between species.(Pybus and Samuel, 1984; Lankester and Hauta, 1989; Lankester and Fong,1989). Thus, available diagnostic techniques cannot routinelydifferentiate between infection with different nematodes.

[0005] Studies directed at identifying antibodies in the serum ofinfected animals have not resulted in reliable diagnostic methods.Although anti-P. tenuis antibodies have been detected in the serum ofelk (wapiti; Cervus elaphus canadensis, Neumann et al., 1994; Bienek etal., 1998) and of goats (Dew et al., 1992), these antibodies areinconsistently detected in the serum of the definitive host, WTD (Dew etal., 1992; Duffy et al., 1993). Furthermore, even in those studies inwhich antibodies were detected, they were not measurable until at least75 days post-exposure. (Duffy et al., supra). In addition, theserological cross-reactivity of anti-P. tenuis antibody against antigensof the other closely related nematodes has not been assessed.

[0006] Thus, there remains a need for early and accurate identificationof parasitic nematode infections. In addition, there is a need foridentification of antigens that specifically and uniquely identifyparasites such as P. tenuis or E. cervi.

SUMMARY OF THE INVENTION

[0007] Described herein are novel common and specific Protostrongylideaantigens, polynucleotides encoding these antigens and antibodies whichrecognize these antigens. Early and accurate diagnostic methods forparasitic infection are disclosed.

[0008] Thus, in one aspect, the invention includes isolated immunogenicProtostrongylidae antigens, for example a P. tenuis-specific 20 kDaantigen, a P. tenuis-specific 37 kDa antigen, an E. cervi-specific 37kDa antigen, a 52 kDa antigen and a P. tenuis-specific 75 kDa antigen, acommon 105 kDa antigen or a common 158 kDa antigen, as determined bySDS-PAGE gel electrophoresis. Thus, antigens specific for P. tenuis orE. cervi are also provided. Exemplary P. tenuis-specific antigensinclude a P. tenuis-specific 20 kDa antigen, a P. tenuis-specific 37 kDaantigen and a P. tenuis-specific 75 kDa antigen, as determined bySDS-PAGE gel electrophoresis. Exemplary E. cervi-specific antigensinclude an E. cervi-specific 37 kDa antigen and an E. cervi-specific 52kDa antigen, as determined by SDS-PAGE gel electrophoresis.

[0009] In another aspect, antibodies that specifically recognize acommon Protostrongylidae antigen are provided. Antibodies thatspecifically recognize P. tenuis-specific or E. cervi-specific antigensare also described.

[0010] In another aspect, the invention provides polynucleotidesencoding common, P. tenuis-specific or E. cervi-specific antigens.

[0011] Methods of diagnosing Protostrongylidae infection in a vertebratesubject by detecting the presence of at least one commonProtostrongylidae antigen in a biological sample, for example a serumsample, obtained from the subject are also provided. In someembodiments, the presence of one common antigen is detected, while inother embodiments, multiple common antigens are detected. Methods ofspecifically diagnosing P. tenuis or E. cervi infection using at leastone P. tenuis-specific or E. cervi-specific antigens are also provided.The common or specific antigens can be detected, for example, usingantibodies, using nucleic acid probes or using PCR. In one embodiment,the common or specific antigens are detected by (a) reacting thebiological sample with one or more isolated common or one or morespecific antigens under conditions which allow anti-Protostrongylidae,P. tenuis or E. cervi antibodies, when present in the sample, tospecifically bind with said common antigens; (b) removing unboundantibodies; (c) providing one or more moieties capable of associatingwith the bound antibodies; and (d) detecting the presence or absence ofthe one or more moieties. The one or more moieties may comprise adetectably labeled immunoglobulin antibody.

[0012] In another aspect, methods of detecting, in a biological sample,antibodies to parasites comprising (a) reacting the biological samplewith an antigen preparation selected from the group consisting of anES-L3 antigen preparation and an sL3 antigen preparation, underconditions which allow parasitic antibodies to bind to an antigen in theantigen preparations and form an antigen:antibody complex; and (b)detecting the presence or absence of said complex are provided. Theparasites may be a member of the Protostrongylidae family or mayspecifically be P. tenuis or E. cervi.

[0013] Kits for use in the diagnostic methods described herein are alsoprovided. The kits comprise, in a suitable packaging, one or more commonor P. tenuis- or E. cervi-specific antigens immobilized on a solidsupport; and a reagent suitable for detecting, in a biological sample,the presence of antibodies to the one or more common or P. tenuis- or E.cervi-specific antigens.

[0014] In yet another aspect, the invention common antigens obtained by(a) providing a cDNA library which expresses protostrongylidae genes;(b) screening the expressed genes of the cDNA library with a source ofanti-protostrongylidae antibodies to identify cDNA clones which expresscommon antigens; and (c) transforming a host cell with the cDNA cloneswhich express the common antigen. Also provided are P. tenuis-specificantigens obtained by (a) providing a cDNA library which expresses P.tenuis genes; (b) screening the expressed genes of the cDNA library witha source of anti-P. tenuis antibodies to identify cDNA clones whichexpress P. tenuis-specific antigens; and (c) transforming a host cellwith the cDNA clones which express the P. tenuis-specific antigen. Alsoprovided are E. cervi-specific antigens obtained by (a) providing a cDNAlibrary which expresses E. cervi genes; (b) screening the expressedgenes of the cDNA library with a source of anti-E. cervi antibodies toidentify cDNA clones which express E. cervi-specific antigens; and (c)transforming a host cell with the cDNA clones which express the E.cervi-specific antigen.

[0015] In another aspect, methods of isolating common or specificantigens are provided. In one embodiment, the antigens are isolated fromexcretory-secretory (ES) products, for example from the third-larvalstage. Alternatively, antigens can be isolated directly from L3 or fromadult organisms.

[0016] These and other embodiments of the subject invention will readilyoccur to those of skill in the art in light of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1, panels A-F, depict serum titers ofanti-Parelaphostrongylus tenuis antibodies in infected white-taileddeer. The legend with each panel refers to the animal by “#” and inparentheses to the number of P. tenuis third-stage larvae (L3) used toinfect the animal. Antibody titers were determined in blood samplestaken at 7 days pre-infection (−7), 21 or 28, 63 or 69 and 141 or 147days post-infection, as shown on the x-axis of each graph.

[0018]FIG. 2 is a graph depicting reactivity of sera fromhelminth-infected cervids against somatic antigens of P. tenuis L3. Serafrom caribou infected with P. andersoni and E. rangiferi (n=1) or E.rangiferi (n=3), red deer infected with E. cervi (n=3) and elk infectedwith Fascioloides magna (n=1) were compared with serum from P. tenuisinfected white-tailed deer (n=6) for reactivity against somatic antigensof P. tenuis L3 (dark bars) by ELISA. Each serum sample was also testedfor reactivity against an irrelevant antigen, bovine serum albumin(light bars). Error bars represent standard deviations from the mean.

[0019]FIG. 3 is a reproduction of a photograph showing immunoblotting ofL3 nematode lysates with sera from infected WTD. The right lane showsunique recognition of a 37 kDa P. tenuis antigen by serum from infectedwhite-tailed deer. Serum samples from caribou with E. rangiferi (Er) orconcurrent E. rangiferi and P. andersoni (Er & Pa) infections werecompared with serum from P. tenuis-infected white-tailed deer (Pt) forrecognition of somatic P. tenuis L3 antigens by immunoblotting. WTDanti-P. tenuis serum uniquely recognized the 37 kDa antigen of P. tenuisL3.

[0020]FIG. 4 shows unique recognition of adult P. tenuis antigens byWhite-tailed deer serum before (pre) and after (post) infection withparasite. Serum obtained from P. tenuis-infected white-tailed deer(post) reacted with a 37 kDa antigen, a 20 kDa antigen, and a 75 kDaantigen present in adult P. tenuis (Pt), but did not react with anyantigen of similar size in the other parasites (Par), e.g., adult E.rangiferi (Er) or E. cervi (Ec). Pre-infection serum (Pre) did notrecognize the specific antigens in P. tenuis, E. rangiferi or E. cervi.

[0021]FIG. 5, panels A-R, are graphs depicting optical density values ofindirect ELISAs using excretory-secretory products of third-stage larvae(ES-L3), somatic antigens of third-stage larvae (L3), and somaticantigens (sA) of adult P. tenuis reacted with anti-P. tenuis antibodiesfrom adult WTD. The heading on each panel refers to the animal (“#”)and, in parentheses, the number and stage of larvae used to infect theanimal. The left column of panels (A-F) shows ES-L3 antigens, the middlecolumn (panels G-L) shows L3 antigens and the right hand column ofpanels (M-R) shows sA antigens The x-axis of each graph shows the days,post-infection, that the serum samples were taken from the infectedanimals. Antibodies against ES-L3 and sL3 increased quickly followinginfection and remained high.

[0022]FIG. 6 depicts early detection of antibody to P. tenuis antigensusing an indirect ELISA.

[0023]FIG. 7 depicts a comparison of antibodies obtained from P. tenuis,P. andersoni, E. rangiferi and E. cervi using an indirect ELISA.

[0024]FIG. 8, panels A-C, are reproductions of photographs of anSDS-PAGE gel immunoblot of P. tenuis antigens obtained from ES-L3 (panelA), sL3 (panel B) or sA (panel C).

[0025]FIG. 9 depicts immunoblot analysis of P. tenuis antigens usingserum obtained from an infected white-tailed deer.

[0026]FIG. 10 is an autoradiograph depicting cDNA synthesized from P.tenuis total RNA.

[0027]FIG. 11 depicts P. tenuis antigen-producing clones. E. coli XLIwas infected with 6×10³ P. tenuis cDNA-UNIZAP phages of the amplifiedlibrary, and grown on an NZY plate. Plaques produced by the lysis ofbacteria were transferred to nitrocellulose and the presence of putativeP. tenuis antigens identified with the antiserum. One plaque (arrow)produced antigen that reacted with anti-P. tenuis antiserum.

[0028]FIG. 12 is a reproduction of a photograph of a nitrocellulosemembrane containing P. tenuis cDNA clones identified from primaryscreening with mouse anti-P. tenuis antiserum were re-screened withother sera/antisera (2. WID anti-P. tenuis, 3. WTD normal serum, 4. Elkanti-Dictyocaulus) to assess cross-reactivity or uniqueness of clones.

[0029]FIG. 13 depicts sizes of various cloned P. tenuis genes encodingantigens. The four right-most lanes show individual clones, labeled byclone number. The fifth lane from the right, labeled V1, show themolecular weight marker. The three left-most lanes, labeled 1′, 2′ and3′, are clones without inserts (controls).

[0030]FIG. 14 shows unique recognition of adult E. cervi (Ec) antigensin red deer serum before (“−”) and after (“+”) infection with parasite.Serum obtained from E. cervi-infected red deer (“+”) reacted with a 52kDa present in E. cervi (Ec), but did not react with any antigen ofsimilar size in the other parasites, e.g., adult E. rangiferi (Er) or P.tenuis (Pt). Pre-infection serum (“−”) did not recognize the specificantigens in P. tenuis, E. rangiferi or E. cervi.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, recombinant DNA technology, and immunology, which arewithin the skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Vols. I, II and III, Second Edition (1989); DNACloning, Vols. I and II (D. N. Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed.1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, B., APractical Guide to Molecular Cloning (1984); the series, Methods InEnzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); andHandbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.Blackwell eds., 1986, Blackwell Scientific Publications).

[0032] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0033] Definitions

[0034] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0035] It must be noted that, as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “a P. tenuis antigen” includes a mixture of two ormore P. tenuis antigens, and the like.

[0036] The term “polypeptide” or “protein” when used with reference toan antigen, such as a P. tenuis-specific or E. cervi-specific antigendescribed herein, refers to polypeptides, whether native, recombinant orsynthetic, which are derived from any of the various parasitic strains,particularly from the family Protostrongylidae. In the case of antigensspecific for P. tenuis, the polypeptide will be derived from a P. tenuisstrain. In the case of antigens specific for E. cervi, the polypeptidewill be derived from a E. cervi strain. The polypeptide need not includethe full-length amino acid sequence of the reference molecule but caninclude only so much of the molecule as necessary in order for thepolypeptide to react with the appropriate antibodies. Thus, only one orfew epitopes of the reference molecule need be present. Furthermore, thepolypeptide may comprise a fusion protein between the full-lengthreference molecule or a fragment of the reference molecule, and anotherprotein that does not disrupt the immunogenicity of the antigenicpolypeptide. It is readily apparent that the polypeptide may thereforecomprise the full-length sequence, fragments, truncated and partialsequences, as well as analogs and precursor forms of the referencemolecule. The term also intends deletions, additions and substitutionsto the reference sequence, so long as the polypeptide retains theability to react with the anti-parasite antibodies.

[0037] In this regard, particularly preferred substitutions willgenerally be conservative in nature, i.e., those substitutions that takeplace within a family of amino acids that are related in their sidechains. Specifically, amino acids are generally divided into fourfamilies: (1) acidic—aspartate and glutamate; (2) basic—lysine,arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar—glycine, asparagine, glutamine, cysteine, serine threonine,tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimesclassified as aromatic amino acids. For example, it is reasonablypredictable that an isolated replacement of leucine with isoleucine orvaline, an aspartate with a glutamate, a threonine with a serine, or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid, will not have a major effect on the biologicalactivity. Proteins having substantially the same amino acid sequence asthe reference molecule, but possessing minor amino acid substitutionsthat do not substantially affect the antibody binding capabilities ofthe protein, are therefore within the definition of the referencepolypeptide.

[0038] An “antigen” refers to any molecule or compound which elicits ahumoral or cell-mediated immune response. As used herein, a “P.tenuis-specific antigen” is any antigen (e.g., polypeptide) derived fromP. tenuis which reacts predominantly with antibodies against P. tenuisbut not with antibodies against other closely related parasiticnematodes of the family Protostrongylidae. Representative P.tenuis-specific antigens include, but are not limited to, a 20 kDaantigen, a 37 kDa antigen (referred to as “a 37 kDa P. tenuis-specificantigen” to distinguish from the E. cervi-specific antigen having thesame apparent molecular weight) and a 75 kDa antigen. Molecular weightsof the antigens are determined using standard gel electrophoresisprotocols, for example as described below in the Examples, 10% SDS pagegels under reducing conditions. Similarly, an “E. cervi-specificantigen” is any antigen that reacts predominantly with antibodiesagainst E. cervi. Representative E. cervi-specific antigens include, butare not limited to, a 37 kDa antigen isolated from third-stage larvae(referred to as “a 37 kDa E. cervi-specific antigen” to distinguish fromthe P. tenuis specific antigen of the same apparent molecular weight)and a 52 kDa antigen. Antigens that react with various members of theProtostrongylidae family (e.g., E. cervi, P. andersoni, E. rangiferi, P.tenuis) are termed “common” antigens. Non-limiting examples of commonantigens include a 105 kDa antigen and 158 kDa antigen.

[0039] By “epitope” is meant a site on an antigen to which specific Bcells and/or T cells respond. The term is also used interchangeably with“antigenic determinant” or “antigenic determinant site.” An epitope cancomprise 3 or more amino acids in a spatial conformation unique to theepitope. Generally, an epitope consists of at least 5 such amino acidsand, more usually, consists of at least 8-10 such amino acids. Methodsof determining spatial conformation of amino acids and conformationalepitopes of a given protein are known in the art and include, forexample, x-ray crystallography and 2-dimensional nuclear magneticresonance. Furthermore, the identification of epitopes in a givenprotein is readily accomplished using techniques well known in the art,such as by the use of hydrophobicity studies and by site-directedserology. See, also, Geysen et al., Proc. Natl. Acad. Sci. USA (1984)81:39984002 (general method of rapidly synthesizing peptides todetermine the location of immunogenic epitopes in a given antigen); U.S.Pat. No. 4,708,871 (procedures for identifying and chemicallysynthesizing epitopes of antigens); and Geysen et al., MolecularImmunology (1986) 23:709-715 (technique for identifing peptides withhigh affinity for a given antibody). Antibodies that recognize the sameepitope can be identified in a simple immunoassay showing the ability ofone antibody to block the binding of another antibody to a targetantigen. See, for example, “Epitope Mapping Protocols in Methods inMolecular Biology,” vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press,Totowa, N.J.

[0040] By “subunit antigen composition” is meant a compositioncontaining at least one immunogenic polypeptide, but not all antigens,derived from or homologous to an antigen from a pathogen of interest.Such a composition is substantially free of intact pathogen cells orparticles, or the lysate of such cells or particles. Thus, a “subunitantigen composition” is prepared from at least partially purified(preferably substantially purified) immunogenic polypeptides from thepathogen, or recombinant analogs thereof. A subunit antigen compositioncan comprise the subunit antigen or antigens of interest substantiallyfree of other antigens or polypeptides from the pathogen.

[0041] A “purified” protein or polypeptide is a protein which isrecombinantly or synthetically produced, or isolated from its naturalhost, such that the amount of protein present in a composition issubstantially higher than that present in a crude preparation. Ingeneral, a purified protein will be at least about 50% homogeneous andmore preferably at least about 80% to 90% homogeneous.

[0042] As used herein, a “biological sample” refers to a sample oftissue or fluid isolated from a subject, including but not limited to,for example, blood, plasma, serum, fecal matter, urine, bone marrow,bile, spinal fluid, lymph fluid, samples of the skin, externalsecretions of the skin, respiratory, intestinal, and genitourinarytracts, samples derived from the gastric epithelium and gastric mucosa,tears, saliva, milk, blood cells, organs, biopsies and also samples ofin vitro cell culture constituents including but not limited toconditioned media resulting from the growth of cells and tissues inculture medium, e.g., recombinant cells, and cell components.

[0043] As used herein, the terms “label” and “detectable label” refer toa molecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes,metal ions, ligands (e.g., biotin or haptens) and the like. The term“fluorescer” refers to a substance or a portion thereof which is capableof exhibiting fluorescence in the detectable range. Particular examplesof labels which may be used under the invention include fluorescein,rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum esters,NADPH and α-β-galactosidase.

[0044] The term “isolated” means separated from constituents, cellularand otherwise, with which the polynucleotide, peptide, polypeptide,protein, antibody or fragments thereof, are normally associated with innature. As is apparent to those of skill in the art, a non-naturallyoccurring polynucleotide, polypeptide, protein, antibody, or fragmentsthereof, does not require isolation to distinguish it from its naturallyoccurring counterpart.

[0045] The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers.

[0046] A polynucleotide is composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T);and uracil (U) for thymine (T) when the polynucleotide is RNA. Thus, theterm polynucleotide sequence is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

[0047] An “isolated polynucleotide” molecule is a nucleic acid moleculeseparate and discrete from the whole organism with which the molecule isfound in nature; or a nucleic acid molecule devoid, in whole or part, ofsequences normally associated with it in nature; or a sequence, as itexists in nature, but having heterologous sequences (as defined below)in association therewith.

[0048] A “vector” is a replicon, such as a plasmid, phage, or cosmid, towhich another DNA segment may be attached so as to bring about thereplication of the attached segment.

[0049] A DNA “coding sequence” or a “nucleotide sequence encoding” aparticular protein, is a DNA sequence which is transcribed andtranslated into a polypeptide in vitro or in vivo when placed under thecontrol of appropriate regulatory elements. The boundaries of the codingsequence are determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxy) terminus. A transcriptiontermination sequence will usually be located 3′ to the coding sequence.

[0050] DNA “control elements” refers collectively to promoters, ribosomebinding sites, polyadenylation signals, transcription terminationsequences, upstream regulatory domains, enhancers, and the like, whichcollectively provide for the transcription and translation of a codingsequence in a host cell. Not all of these control sequences need alwaysbe present in a recombinant vector so long as the desired gene iscapable of being transcribed and translated.

[0051] “Operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter and the coding sequence and the promoter canstill be considered “operably linked” to the coding sequence.

[0052] A control element, such as a promoter, “directs thetranscription” of a coding sequence in a cell when RNA polymerase willbind the promoter and transcribe the coding sequence into mRNA, which isthen translated into the polypeptide encoded by the coding sequence.

[0053] A “host cell” is a cell which has been transformed, or is capableof transformation, by an exogenous nucleic acid molecule.

[0054] A cell has been “transformed” by exogenous DNA when suchexogenous DNA has been introduced inside the cell membrane. ExogenousDNA may or may not be integrated (covalently linked) into chromosomalDNA making up the genome of the cell. In procaryotes and yeasts, forexample, the exogenous DNA may be maintained on an episomal element,such as a plasmid. With respect to eucaryotic cells, a stablytransformed cell is one in which the exogenous DNA has become integratedinto the chromosome so that it is inherited by daughter cells throughchromosome replication. This stability is demonstrated by the ability ofthe eucaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

[0055] “Homology” refers to the percent identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown in the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs such as ALIGN, Dayhoff, M. O. (1978) in Atlas ofProtein Sequence and Structure 5:Suppl. 3, National biomedical ResearchFoundation, Washington, D.C. Preferably, default parameters are used foralignment. One alignment program is BLAST, used with default parameters.For example, BLASTN and BLASTP can be used using the following defaultparameters: genetic code=standard; filter=none; strand=both; cutoff=60;expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGHSCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the following internet address:http://www.ncbi.nlm.gov/cgi-bin/BLAST.

[0056] Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. Two DNA,or two polypeptide sequences are “substantially homologous” to eachother when the sequences exhibit at least about 80%-85%, preferably atleast about 90%, and most preferably at least about 95%-98% sequenceidentity over a defined length of the molecules, as determined using themethods above. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence. DNA sequences that are substantially homologous can beidentified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic AcidHybridization, supra.

[0057] A “heterologous” region of a DNA construct is an identifiablesegment of DNA within or attached to another DNA molecule that is notfound in association with the other molecule in nature. Thus, when theheterologous region encodes a bacterial gene, the gene will usually beflanked by DNA that does not flank the bacterial gene in the genome ofthe source bacteria. Another example of the heterologous coding sequenceis a construct where the coding sequence itself is not found in nature(e.g., synthetic sequences having codons different from the nativegene). Allelic variation or naturally occurring mutational events do notgive rise to a heterologous region of DNA, as used herein.

[0058] By “vertebrate subject” is meant any member of the subphylumchordata, particularly mammals, including, without limitation, cervids,ruminants, humans, and primates. For example, the term includes reddeer, white tailed deer, elk, moose, caribou and the like. The term doesnot denote a particular age. Thus, both adult and newborn individualsare intended to be covered.

[0059] General Overview

[0060] The present invention provides, in one aspect, novel parasiticantigens (e.g.,antigens from nematodes of the family protostrongylid)and fragments thereof. These antigens are useful in methods ofdiagnosing protostrongylidae infection in general.

[0061] The invention also provides, in another aspect, antigens whichspecifically and uniquely recognize Parelaphostrongylus tenuis (P.tenuis) infection. These P. tenuis-specific antigens can be used todistinguish P. tenuis infection from infection with other parasiticmembers of the Protostrongylidae family, for example E. cervi, E.rangiferi, P. ocoilei and P. andersoni. Antigens which specifically anduniquely recognize E. cervi infection are also provided. These E.cervi-specific antigens can be used to distinguish E. cervi infectionfrom infection with other parasitic members of the Protostrongylidaefamily, for example P. tenuis, E. rangiferi, P. ocoilei and P.andersoni. Antibodies to these antigens and genes (e.g., nucleic acids)encoding the P. tenuis- or E. cervi-specific antigens are also provided.

[0062] The antigens and fragments thereof, antibodies thereto, and genescoding thereof, are useful as diagnostic reagents to detect the presenceof infection in a vertebrate subject, for example, by testing abiological sample (blood, feces, etc.) obtained from these animals forthe presence of these molecules. The presence or absence of antigens canbe detected by a variety of methods, including, for example, use ofantibodies, gel electrophoresis, ELISA, PCR, nucleic acid hybridization,or the like. For example, antibodies can be detected by reacting theputative antibody-containing sample with a parasitic antigen identifiedherein under conditions suitable for forming an antigen:antibodycomplex. The presence of such a complex is indicative of infection.Similarly, nucleic acid probes or primers specific for the genesencoding the antigen(s) of interest can be isolated or synthesized andthe sample reacted under conditions such that the probe specificallyhybridizes to the target sequence (or amplifies a PCR product). Thus,the invention includes methods of specifically diagnosing P. tenuis orE. cervi infection, by detecting the presence of P. tenuis- or E. cervispecific antigens and antibodies as well as diagnosing protostrongylidaeinfection in general. Thus, specific or common antigens (i.e., antigensthat react with various members of the Protostrongylidae family) can beused to diagnose parasitic infection. It is to be understood that suchdiagnostic assays can be conducted using one or more common antigens,one or more specific antigens, or, alternatively, may use a combinationof common and specific antigens.

[0063] In yet another aspect, the genes encoding the antigens can becloned and used to design probes to detect and isolate homologous genesin other bacterial strains. For example, fragments comprising at leastabout 8-100 nucleotides, more preferably at least about 10-50nucleotides, and most preferably about 12-30 or more nucleotides, willfind use in these embodiments.

[0064] Antigens

[0065] The antigens described herein and active fragments and analogsderived from the same, can be produced by a variety of methods. In onemethod, Protostrongylidae-common antigens and P. tenuis- or E.cervi-specific antigens can be isolated directly from the organisms(e.g., nematodes) which express the same. As described in detail below,the life cycle of these organisms are quite complex, involving larvaland adult stages. Suitable sources (e.g., stages) for the novel antigensare disclosed herein and can be readily determined by one of skill inthe art in view of the teachings of this specification. For example, inone aspect, antigens can be isolated from the excretory-secretoryproducts of the third larval stage (ES-L3) or directly from L3 or adultorganisms. Generally, antigens (both common and specific) can beisolated by first preparing a crude extract which lacks cellularcomponents and several extraneous proteins. The desired antigens canthen be further purified, i.e, by column chromatography, HPLC,immunoadsorbent techniques or other conventional methods well known inthe art. The antigens may be tested for specificity, for example asdescribed in the Examples, by determining whether they bind toantibodies present in the serum of infected subjects. Once isolated, theamino acid sequence of the antigens can be readily determined, forexample by cleavage and identification of the amino acid residues.

[0066] Alternatively, the antigens can be recombinantly produced. Theserecombinant products can take the form of partial protein sequences,full-length sequences, precursor forms that include signal sequences,mature forms without signals, or even fusion proteins (e.g., with anappropriate leader for the recombinant host, or with another antigenicsequence for P. tenuis or another pathogen).

[0067] The antigen-encoding genes of the present invention can beisolated using standard techniques, well known in the art. For example,gene libraries can be constructed and the resulting clones used totransform an appropriate host cell. Colonies can be pooled and screenedusing polyclonal serum or monoclonal antibodies to the P. tenuis or E.cervi antigen in question. As described in the Examples, cDNA clones canbe prepared from mRNA of the parasite and these clones used to producepolypeptides which can be screened for their reactivity with antibodies.Both common and E. cervi- or P. tenuis-specific clones can be identifiedthis way. Antigen-encoding genes of between about 500 and about 1,500base pairs in size have been identified (see, Example 3).

[0068] Alternatively, once the amino acid sequences are determined,oligonucleotide probes which contain the codons for a portion of thedetermined amino acid sequences can be prepared and used to screengenomic or cDNA libraries for genes encoding the subject proteins. Thebasic strategies for preparing oligonucleotide probes and DNA libraries,as well as their screening by nucleic acid hybridization, are well knownto those of ordinary skill in the art. See, e.g., DNA Cloning: Vol. I,supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis,supra; Sambrook et al., supra. Once a clone from the screened libraryhas been identified by positive hybridization, it can be confirmed byrestriction enzyme analysis and DNA sequencing that the particularlibrary insert contains a P. tenuis gene or a homolog thereof. The genescan then be further isolated using standard techniques and, if desired,PCR approaches or restriction enzymes employed to delete portions of thefull-length sequence. Similarly, genes can be isolated directly frombacteria using known techniques, such as phenol extraction and thesequence further manipulated to produce any desired alterations. See,e.g., Sambrook et al., supra, for a description of techniques used toobtain and isolate DNA.

[0069] Alternatively, DNA sequences encoding the proteins of interestcan be prepared synthetically rather than cloned. The DNA sequences canbe designed with the appropriate codons for the particular amino acidsequence. In general, one will select preferred codons for the intendedhost if the sequence will be used for expression. The complete sequenceis assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge(1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay etal. (1984) J. Biol. Chem. 259:6311.

[0070] Once coding sequences for the desired proteins have been preparedor isolated, they can be cloned into any suitable vector or replicon.Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra; B.Perbal, supra.

[0071] The gene can be placed under the control of a promoter, ribosomebinding site (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired protein is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. If a signal sequence is included, it caneither be the native, homologous sequence, or a heterologous sequence.Leader sequences can be removed by the host in post-translationalprocessing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.

[0072] Other regulatory sequences may also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Regulatory sequences are known to those of skill inthe art, and examples include those which cause the expression of a geneto be turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound. Other types ofregulatory elements may also be present in the vector, for example,enhancer sequences.

[0073] The control sequences and other regulatory sequences may beligated to the coding sequence prior to insertion into a vector, such asthe cloning vectors described above. Alternatively, the coding sequencecan be cloned directly into an expression vector which already containsthe control sequences and an appropriate restriction site.

[0074] In some cases it may be necessary to modify the coding sequenceso that it may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. It may also bedesirable to produce mutants or analogs of the P. tenuis antigen inquestion. Mutants or analogs may be prepared by the deletion of aportion of the sequence encoding the protein, by insertion of asequence, and/or by substitution of one or more nucleotides within thesequence. Techniques for modifying nucleotide sequences, such assite-directed mutagenesis, are described in, e.g., Sambrook et al.,supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

[0075] The expression vector is then used to transform an appropriatehost cell. A number of mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Similarly,bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcusspp., will find use with the present expression constructs. Yeast hostsuseful in the present invention include inter alia, Saccharomycescerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha,Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii,Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.Insect cells for use with baculovirus expression vectors include, interalia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugperda, and Trichoplusia ni.

[0076] Other systems for expression of the desired antigens includeinsect and plant cells and vectors suitable for use in these cells. Thesystems most commonly used are derived from the baculovirus Autographacalifornica polyhedrosis virus (AcNPV). Generally, expression in insectcells is achieved by using a bacterial plasmid which contains a fragmentof the baculovirns genome and an insertion site for the heterologousgene (e.g., encoding the epitope of interest). Sufficient wild-typebacoluvirus sequence is also included both that the plasmid vector (andtransgene) homologous recombine into the baculovirus genome.

[0077] Promoters for use in the vectors are typically derived fromstructural genes, abundantly transcribed at late times in a viralinfection cycle. Examples include sequences derived from the geneencoding the viral polyhedron protein, Friesen et al., (1986) “TheRegulation of Baculovirus Gene Expression” in: The Molecular Biology ofBaculoviruses (ed. Walter Doerfler); EP Publication Nos. 127,839 and155,476; and the gene encoding the p10 protein Vlak et al., J. Gen.Virol. (1988) 69:765. The plasmid usually also contains the polyhedrinpolyadenylation signal (Miller et al., Ann. Rev. Microbiol. (1988)42:177) and aprocaryotic ampicillin-resistance (amp) gene and origin ofreplication for selection and propagation in E. coli. DNA encodingsuitable signal sequences can also be included and is generally derivedfrom genes for secreted insect or baculovirus proteins, such as thebaculovirns polyhedrin gene (Carbonell et al., Gene (1988) 73:409), aswell as mammalian signal sequences such as those derived from genesencoding human α-interferon, Maeda et al., Nature (1985) 315:592; humangastrin-releasing peptide, Lebacq-Verheyden et al., Molec. Cell. Biol.(1988) 8:3129; human IL-2, Smith et al., Proc. Natl. Acad. Sci. USA(1985) 82:8404; mouse IL-3, (Miyajima et al., Gene (1987) 58:273; andhuman glucocerebrosidase, Martin et al., DNA (1988) 7:99.

[0078] Depending on the expression system and host selected, theproteins of the present invention are produced by culturing host cellstransformed by an expression vector described above under conditionswhereby the protein of interest is expressed. The protein is thenisolated from the host cells and purified. If the expression systemsecretes the protein into the growth media, the protein can be purifieddirectly from the media. If the protein is not secreted, it is isolatedfrom cell lysates. The selection of the appropriate growth conditionsand recovery methods are within the skill of the art.

[0079] The proteins of the present invention may also be produced bychemical synthesis such as solid phase peptide synthesis, using knownamino acid sequences or amino acid sequences derived from the DNAsequence of the genes of interest. Such methods are known to thoseskilled in the art. See, e.g., J. M. Stewart and J. D. Young, SolidPhase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill.(1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis,Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, AcademicPress, New York, (1980), pp. 3-254, for solid phase peptide synthesistechniques; and M. Bodansky, Principles of peptide Synthesis,Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., ThePeptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classicalsolution synthesis. Chemical synthesis of peptides may be preferable ifa small fragment of the antigen in question is desired.

[0080] Antibodies

[0081] The protostrongylidae (e.g., P. tenuis-specific, E.cervi-specific and common antigens) of the present invention, or theirfragments, can be used to produce antibodies, both polyclonal andmonoclonal. If polyclonal antibodies are desired, a selected mammal,(e.g., mouse, rabbit, goat, horse, etc.) is immunized with an antigen ofthe present invention, or its fragment, or a mutated antigen. Serum fromthe immunized animal is collected and treated according to knownprocedures. See, e.g., Jurgens et al. (1985) J. Chrom. 348:363-370. Ifserum containing polyclonal antibodies is used, the polyclonalantibodies can be purified by immunoaffinity chromatography, using knownprocedures.

[0082] Monoclonal antibodies to the common and specific antigens and tothe fragments thereof, can also be readily produced by one skilled inthe art. The general methodology for making monoclonal antibodies byusing hybridoma technology is well known. Immortal antibody-producingcell lines can be created by cell fusion, and also by other techniquessuch as direct transformation of B lymphocytes with oncogenic DNA, ortransfection with Epstein-Barr virus. See, e.g., M. Schreier et al.,Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodiesand T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies(1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783;4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890.Panels of monoclonal antibodies produced against the P. tenuis antigensor fragments thereof, can be screened for various properties; i.e., forisotype, epitope, affinity, etc. Monoclonal antibodies are useful inpurification, using immunoaffinity techniques, of the individualantigens which they are directed against. Both polyclonal and monoclonalantibodies are also useful for diagnostic purposes.

[0083] Diagnostic Applications

[0084] As explained above, the antigens of the present invention may beused as diagnostics to detect the presence of reactive antibodies in abiological sample in order to determine the presence of infection. Inparticular, the antigens are used herein as diagnostics to detect thepresence of reactive antibodies directed against the worms in abiological sample. Furthermore, antigens specific for certain nematodes(e.g., P. tenuis or E. cervi) can be used to differentiate betweeninfection with members of the Protostrongylidae family. For example, thepresence of antibodies reactive with the cross-reactive and/or thespecific antigens can be detected using standard electrophoretic andimmunodiagnostic techniques, including immunoassays such as competition,direct reaction, or sandwich type assays. Such assays include, but arenot limited to, Western blots; agglutination tests; enzyme-labeled andmediated immunoassays, such as ELISAS; biotinlavidin type assays;radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. Thereactions generally include revealing labels such as fluorescent,chemiluminescent, radioactive, enzymatic labels or dye molecules, orother methods for detecting the formation of a complex between theantigen and the antibody or antibodies reacted therewith.

[0085] The aforementioned assays generally involve separation of unboundantibody in a liquid phase from a solid phase support to whichantigen-antibody complexes are bound. Solid supports which can be usedin the practice of the invention include substrates such asnitrocellulose (e.g., in membrane or microtiter well form);polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex(e.g., beads or microtiter plates); polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

[0086] Typically, a solid support is first reacted with a solid phasecomponent (e.g., one or more common or specific antigens) under suitablebinding conditions such that the component is sufficiently immobilizedto the support. Sometimes, immobilization of the antigen to the supportcan be enhanced by first coupling the antigen to a protein with betterbinding properties. Suitable coupling proteins include, but are notlimited to, macromolecules such as serum albumins including bovine serumalbumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art. Other molecules that can be used to bind the antigens to thesupport include polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and the like. Suchmolecules and methods of coupling these molecules to the antigens, arewell known to those of ordinary skill in the art. See, e.g., Brinldey,M. A. Bioconjugate Chem. (1992) 3:2-13; Hashida et al., J. Appl.Biochem. (1984) 6:56-63; and Anjaneyulu and Staros, International J. ofPeptide and Protein Res. (1987) 30:117-124.

[0087] After reacting the solid support with the solid phase component,any non-immobilized solid-phase components are removed from the supportby washing, and the support-bound component is then contacted with abiological sample suspected of containing ligand moieties (e.g.,antibodies against the immobilized antigens) under suitable bindingconditions. After washing to remove any non-bound ligand, a secondarybinder moiety is added under suitable binding conditions, wherein thesecondary binder is capable of associating selectively with the boundligand. The presence of the secondary binder can then be detected usingtechniques well known in the art.

[0088] More particularly, an ELISA method can be used, wherein the wellsof a microtiter plate are coated with an antigen of interest, forexample an antigen common to various species of protostrongylidae or P.tenuis- or E. cervi-specific antigens). A biological sample containingor suspected of containing anti-antigen immunoglobulin molecules is thenadded to the coated wells. After a period of incubation sufficient toallow antibody binding to the immobilized antigen, the plate(s) can bewashed to remove unbound moieties and a detectably labeled secondarybinding molecule added. The secondary binding molecule is allowed toreact with any captured sample antibodies, the plate washed and thepresence of the secondary binding molecule detected using methods wellknown in the art.

[0089] Thus, in one particular embodiment, the presence of boundanti-Protostrongylid antigen ligands from a biological sample can bereadily detected using a secondary binder comprising an antibodydirected against the antibody ligands. For example, E. cervi- or P.tenuis-specific antigens can be used to detect antibodies in a samplewith the aid of appropriate anti-species immunoglobulins (Ig). A numberof anti-human immunoglobulin (Ig) molecules are known in the art (e.g.,commercially available goat anti-human Ig or rabbit anti-human Ig). Igmolecules for use herein will preferably be of the IgG or IgA type,however, IgM may also be appropriate in some instances. The Ig moleculescan be readily conjugated to a detectable enzyme label, such ashorseradish peroxidase, glucose oxidase, Beta-galactosidase, alkalinephosphatase and urease, among others, using methods known to those ofskill in the art. An appropriate enzyme substrate is then used togenerate a detectable signal. In other related embodiments,competitive-type ELISA techniques can be practiced using methods knownto those skilled in the art.

[0090] Assays can also be conducted in solution, such that the P. tenuisantigens and antibodies specific for those antigens form complexes underprecipitating conditions. In one particular embodiment, one or morecommon or specific antigens can be attached to a solid phase particle(e.g., an agarose bead or the like) using coupling techniques known inthe art, such as by direct chemical or indirect coupling. Theantigen-coated particle is then contacted under suitable bindingconditions with a biological sample suspected of containing antibodiesdirected against P. tenuis, E. cervi or other protostrongylidae species.Cross-linking between bound antibodies causes the formation ofparticle-antigen-antibody complex aggregates which can be precipitatedand separated from the sample using washing and/or centrifugation. Thereaction mixture can be analyzed to determine the presence or absence ofantibody-antigen complexes using any of a number of standard methods,such as those immunodiagnostic methods described above.

[0091] In yet a further embodiment, an immunoaffinity matrix can beprovided, wherein a polyclonal population of antibodies from abiological sample suspected of containing anti-P. tenuis (or anti-E.cervi, anti-common or mixtures thereof) antigen molecules is immobilizedto a substrate. In this regard, an initial affinity purification of thesample can be carried out using immobilized antigens. The resultantsample preparation will thus only contain anti-P. tenuis (or anti-E.cervi, anti-common or mixtures thereof) moieties, avoiding potentialnonspecific binding properties in the affinity support. A number ofmethods of immobilizing immunoglobulins (either intact or in specificfragments) at high yield and good retention of antigen binding activityare known in the art. Not being limited by any particular method,immobilized protein A or protein G can be used to immobilizeimmunoglobulins.

[0092] Accordingly, once the immunoglobulin molecules have beenimmobilized to provide an immunoaffinity matrix, labeled P. tenuisantigens (or anti-E. cervi antigens or anti-common antigens or mixturesthereof) are contacted with the bound antibodies under suitable bindingconditions. After any non-specifically bound antigen has been washedfrom the immunoafnnity support, the presence of bound antigen can bedetermined by assaying for label using methods known in the art.

[0093] Additionally, antibodies raised to the antigens, rather than theantigens themselves, can be used in the above-described assays in orderto detect the presence of proteins in a given sample. These assays areperformed essentially as described above and are well known to those ofskill in the art.

[0094] In another embodiment, the presence of antigens (e.g. common orE. cervi- or P. tenuis-specific) can be detected at the nucleic acidlevel. In assaying for the presence of sequences encoding common orspecific antigens, the nucleic acids of the biological sample are firstextracted according to standard methods in the art. For instance, DNAcan be isolated from a biological sample by ethanol precipitation andrepeated phenol:chloroform extractions (see, e.g., Sambrook et al,supra). Alternatively, mRNA can be isolated using various lytic enzymesor chemical solutions according to procedures set forth in Sambrook etal. (1989), supra or extracted by nucleic acid-binding resins followingthe instructions provided by the manufacturer. The mRNA of the antigenof interest contained in the sample is then detected by hybridization(e.g., Northern blot analysis) and/or amplification procedures accordingto the methods known in the art and described herein.

[0095] Nucleic acid molecules having at least 10 nucleotides, preferablyat least about 12 to at least about 25, and more preferably at leastabout 15 to at least about 20 nucleotides, and exhibiting sequencecomplementarity or homology to the common or specific antigens describedherein find utility as hybridization probes. It is know that a“perfectly matched” probe is not needed for specific hybridization.Minor changes in probe sequence achieved by substitution, deletion orinsertion of a small number of bases do not affect the hybridizationspecificity. In general, as much as 20% base-pair mismatch (whenoptimally aligned) can be tolerated. Preferably, a probe useful fordetecting the aforementioned antigen of interest is at least 80%identical to the homologous region of the target sequence, morepreferably at least about 85% identical and even more preferably atleast about 90% identical.

[0096] These probes can be used in radioassays (e.g., Southern andNorthern blot analysis) to detect, prognose, diagnose or monitor variousconditions and symptoms resulting from Protostrongylidae infection. Thetotal size of the fragment, as well as the size of the complementarystretches, will depend on the intended use or application of theparticular nucleic acid segment. Smaller fragments derived from theknown sequences will generally find use in hybridization embodiments,wherein the length of the complementary region may be varied, such asbetween about 10 and about 100 nucleotides or even longer if preferred.Thus, probes having complementary sequences over stretches greater thanabout 10 nucleotides in length are generally preferred, so as toincrease stability and selectivity of the hybrid, and thereby improvethe specificity of the particular hybrid molecules obtained. Suchfragments may be readily prepared, for example, directly synthesizingthe fragment by chemical means, by application of nucleic acidreproduction technology, such as PCR technology with two primingoligonucleotides as described in U.S. Pat. No. 4,603,102, or byrecombinant means. A preferred probe is about 25 to about 50, morepreferably from about 50 to about 75 or even more preferably, about 50to about 100 nucleotides in length.

[0097] In certain embodiments, it may be advantageous to employ nucleicacid sequences of the present invention in combination with appropriatemeans, such as a label, for detecting hybridization. A wide variety ofappropriate indicator means are available, for example, fluorescent,radioactive, enzymatic or other ligands, such as biotin/avidin. In thecase of enzyme tags, colorimetric indicator substrates are known toprovide a detectable (by eye or by spectrophotometer) signal.

[0098] Hybridization reactions can be performed under conditions ofdifferent “stringency.” Relevant parameters which affect stringencyinclude temperature, ionic strength, time of incubation, the presence ofadditional solutes in the reaction mixture such as formamide, and thewashing procedure. Higher stringency conditions are those conditions,such as high temperature and lower sodium ion concentration, whichrequire higher minimum complementarity between hybridizing elements fora stable complex to form. Conditions that affect stringency are widelyknown, for example as described in Sambrook et al, supra.

[0099] Nucleotide probes may also be used as primers for the detectionof genes in a sample. Amplification of target sequences can be performedby any method employing a primer-dependent polymerase capable ofreplicating a target sequence with reasonable fidelity, for example,natural or recombinant DNA polymerases such as T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase.

[0100] A preferred amplification method is PCR. General procedures forPCR are described, for example, in MacPherson et al., PCR: A PracticalApproach, (IRL Press at Oxford University Press (1991)). PCR conditionsfor individual reactions can be empirically determined. A number ofparameters influence the success of a PCR reaction. Among them areannealing temperature and time, extension time, Mg²⁺ ATP concentration,pH, and the relative concentration of primers, templates anddeoxyribonucleotides. See, e.g., U.S. Pat. Nos. 4,683,202; 4,683,195;4,965,188 and 4,800,159 for a description of the PCR technique.

[0101] Alternatively, the probes can be attached to a solid support foruse in high throughput screening assays using methods known in the art.U.S. Pat. Nos. 5,405,783; 5,412,087 and 5,445,934, for example, disclosethe construction of high density oligonucleotide chips which can containone or more of the sequences for common or specific antigens.

[0102] After amplification, the resulting DNA fragments can be detectedby agarose gel electrophoresis followed by visualization with ethidiumbromide staining and ultraviolet illumination. A specific amplificationof common or specific antigens can be verified by demonstrating that theamplified DNA fragment has the predicted size, exhibits the predictedrestriction digest pattern, and/or hybridizes to the correct cloned DNAsequence.

[0103] The above-described assay reagents, including, for example, theP. tenuis antigens, or antibodies thereto, can be provided in kits, withsuitable instructions and other necessary reagents, in order to conductimmunoassays as described above. The kit can also contain, depending onthe particular immunoassay used, suitable labels and other packagedreagents and materials (i.e. wash buffers and the like). Standardimmunoassays, such as those described above, can be conducted usingthese kits.

[0104] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

EXAMPLES Example 1 Serological Diagnosis of Parelaphostronglyus TenuisInfection and Identification of a Unique P. tenuis Antigen

[0105] 1.A. Materials and Methods

[0106] 1.A.1. Parasites and Sera

[0107] Infective third-stage larvae (L3) of P. tenuis were obtained bypepsin-HCl digestion of snails (Triodopsis multilineata) that had beenexposed to the first-stage larvae (L1) of the parasite 8-16 weeksearlier. The L1 were extracted from the feces of P. tenuis-infectedwhite tailed deer (WTD) from Grand Marais, Minn., USA (47°41′N, 90°35′W)(Forrester and Lankester, 1997). The L3 were used to infect WTD or toprepare somatic antigens. Adult P. tenuis were obtained at postmortemfrom the meninges of the central nervous system of experimentallyinfected WTD (Slomke et al., 1995). Adult Elaphostrongylus rangiferiwere obtained from the chest and thigh muscles of four wild woodlandcaribou (Rangifer tarandus caribou) on the Avalon Peninsula,Newfoundland, Canada (46°47′N 54°10′W). One of the caribou also hadadult P. andersoni in its longissimus dorsi muscles. Serum samples wereobtained from all caribou. Adult E. cervi were obtained from the looseconnective tissue of the skeletal muscles in the axilla and inguinalregions of three red deer (Cervus elaphus elaphus) experimentallyinfected approximately six months prior with E. cervi L3 (Gajadhar etal., 1994). Serial serum samples were obtained from these red deerduring the course of the infection. Serum samples were also obtainedfrom an elk captured from Elk Island National Park, Alberta, Canada,observed to be shedding trematode eggs, and found at postmortem to haveFascioloides magna. All animals were euthanised with an overdose ofxylazine hydrochloride (Rompun, Miles, Etobicoke, Ontario, Canada)followed by T61 (Hoechst, Montreal, Quebec, Canada).

[0108] 1.A.2. Infection of White-tailed Deer with P. tenuis and BloodCollection

[0109] WTD used in the study were acquired as week-old orphaned fawnsand raised in captivity in Saskatchewan where P. tenuis does not occur(Anderson, 1992). The fawns were bottle-fed and maintained in an openpaddock and before transportation to Thunder Bay, Ontario where theywere held on clean concrete. All animals were 9.5 months old at thestart of the experiment. For purposes of infection and blood collection,animals were anaesthetized with Rompun administered using a blowpipe andlightweight syringe. Two WTD were each orally inoculated with six P.tenuis larvae, two were given 20, and two were given between 100 and150. Blood samples were collected from each animal 1 wk beforeinoculation and at different times until the end of the experiment at147 day post-inoculation (dpi). All 6 WTD passed small numbers ofDictyocaulus larvae in their feces during the experiment but no adultspecimens were recovered at necropsy. Two additional WTD were kept inSaskatchewan as uninfected, control animals. Maintenance and use of allexperimental animals were according to the guidelines of the CanadianCouncil on Animal Care.

[0110] 1.A.3. Somatic Parasite Antigens

[0111] Adult and L3 of P. tenuis and adults of E. rangiferi and E. cerviwere washed in PBS, kept on ice and sonicated at 300 W, 1 min at a timefor a total of 5 min or until no discernible worm fragments weremicroscopically visible. The parasite homogenate was centrifuged at10,000 g for 5 min and the supernatant removed and stored at −20° C.until use. Protein concentrations of antigens were determined using theBCA kit (Pierce, Rockford, Ill., USA).

[0112] 1.A.4. ELISA

[0113] Somatic antigens of P. tenuis L3 were diluted in PBS, pH 7.4,adsorbed to wells of microtitre plates (Immulon-4, Dynatech Lab,Chantilly, Va., USA) at 0.4 μg/well and incubated at 37° C. overnight.Unadsorbed proteins were washed off the wells with PBS containing 0.5%Tween 20 (PBST) using a plate washer. Predetermined optimal dilution(1:200) of WTD, caribou and elk sera were made in PBST, and applied at50 μl/well after which the plates were incubated at 37° C. for 3 h. Atthe end of the incubation, plates were washed and alkalinephosphatase-labeled rabbit WTD IgG (Kirkegaard and Perry Laboratories,Gaithersburg, Md., USA), demonstrated to strongly cross-react withcaribou and elk antibodies, was diluted in PBST (1:3,000) and applied at50 μl/well. After a further 2 hour incubation at 37° C., wells of theplate were washed with PBST and phosphatase substrate (p-nitrophenylphosphatase, Kirkegaard and Perry Laboratories, Gaithersburg, Md., USA)added. Color development was allowed to proceed for 1 hr in the dark,stopped by the addition of 5% ethylene diamine tetraacetic acid (EDTA)and measured at a wavelength of 405 nm with a spectrophotometer(Titertek Multiskan, Labsystems, Finland). ELISA results are shown asantibody titers or optical density (OD) values.

[0114] 1.A.5. Immunoblotting

[0115] Somatic antigens of L3 or adult parasites were separated on a 10%SDS-Polyacrylamide gel at 160 V for 45 min, under reducing conditions.The separated proteins were transferred onto nitrocellulose membrane(BioRad, Hercules, Calif.) for immuno-staining. Membranes bearing theproteins were blocked with PBS containing 5% milk (milk-PBS). Deer orcaribou sera were diluted 1 in 25 in 2.5% milk-PBS containing 0.1% Tween20 (milk-PBST), applied to membranes and incubated overnight at roomtemperature. After incubation, the membranes were washed with PBST,following which alkaline phosphatase-labelled rabbit anti-deer IgGdiluted 1 in 500 in milk-PBST was added. After 4 hours of incubation,the membrane was washed three times in PBST followed by a final wash inPBS. Color development reagent (BCIP/NBT phosphatase substrate,Kirkegaard and Perry Laboratories, Gaithersburg, Md.) was added to themembrane and incubated in the dark. The development was stopped after 30min by the addition of tap water to the membrane. Bands detected byimmunoblotting were subjected to densitometric analysis by scanning(Sharp Scanner, Mahwah, N.J., USA) onto a computer equipped with theImageMaster software (Pharmacia, Montreal, Canada).

[0116] 1.A.6. Mathematical and Statistical Analyses

[0117] A regression analysis of optical density values against thereciprocal values of the dilutions were used to generate a straight lineequation from which antibody titers were calculated (Lehtonen andViljanen, 1982). Pre-infection antibody titers were arbitrarily set at 1in 10. Correlations between dependent and independent variables wereassessed by linear regression. Total antibody titers were calculated asthe area under the curve generated by plotting the titers at thedifferent bleeding times. Statistical significances were analyzed at the95% confidence limits. All analyses were performed with the Prismcomputer program (GraphPad software, San Diego, Calif., USA).

[0118] 1.B. Results

[0119] 1.B.1. Anti-P. tenuis L3 Antibodies in Infected White-tailed Deer

[0120] Serum samples collected from six white-tailed deer infected with6, 20 or 100-150 P. tenuis L3 (two animals per dose) containedantibodies that reacted against somatic antigens of P. tenuis L3 inELISA. Anti-P. tenuis antibodies were detected within the first month ofinfection in all animals (FIG. 1), including two animals exposed to asfew as six L3 of P. tenuis and from which only 3 adult worms wererecovered. This early detection indicates that serological testing todetect P. tenuis infection can be used earlier than the currently usedBaermann technique since none of the infected animals passed L1 in thefeces before 90 dpi.

[0121] Early antibody titers ranged from 1 in 70 to 1 in 2830 while peakantibody titers ranged from 1 in 70 to 1 in 5700. In comparison,antibody titers of both un-infected, control animals were less than 1 in10. Animals infected with higher numbers of parasites tended to producehigher peaks of anti-parasite antibodies ®=0.88; p<0.05) and highertotal antibody levels ®=0.85; p<0.05). At necropsy, 3 adult P. tenuiswere recovered from each of 2 WTD (#1 and 2) given doses of 6 L3 each, 7and 14 from the animals (#3 and 4) each given 20 L3, and 52 and 32 fromthe animals (#5 and 6) each given 100-150 L3. In all infected animals,anti-P. tenuis antibody titers persisted throughout the course of theexperiment.

[0122] Such a high sensitivity is important since WTD with natural P.tenuis infections usually have less than 10 adult worms in the cranium,even in enzootic areas (Anderson, 1992). Slomke et al. (1995) reported alow level of infection with a mean number of 3.2 adult worms (max 13)among 311 WTD. The number of adult worms harbored by older animals(i.e., 2-6 years and 7-15 years) was not statistically higher than thatof younger animals (i.e., ≦one year old and younger) suggesting that WTDin enzootic areas ingest a small number of P. tenuis L3 in their firstyear and do not acquire additional P. tenuis afterwards. This isevidence for the existence of concomitant immunity in P. tenuisinfection: WTD exposed to, and harboring P. tenuis worms are protectedagainst the establishment of a newly acquired L3. Re-exposure to theparasite, even when this does not result in a new infection, should helpboost the antibody response and make sero-diagnosis of P. tenuisinfection more likely in enzootic areas.

[0123] In four out of the six WTD, antibody levels peaked between day 21and 69 and then decreased toward the end of the experiment while in theremaining two animals, the highest levels of antibodies amongst the WTDwere observed at the end of the experiment.

[0124] As expected, anti-L3 antibodies persisted throughout the courseof infection in all animals. Without being bound by one theory, thisappears to be due to the cross-reactivity between certain L3 and matureparasite antigens. The persistence of anti-L3 antibodies in WTD after L3have developed into adults, evidences that certain antigens might beshared between L3 and later stages of the parasite. The longevity ofantibodies may also contribute to their persistence in the infectedanimals. A serological test utilizing only a unique antigen present inboth L3 and adult should show a progressive increase in anti-parasitetiters in infected animals.

[0125] Serum samples from caribou infected with E. rangiferi orconcurrently with P. andersoni, and from red deer infected with E. cervialso reacted strongly against somatic L3 antigens with intensitiessimilar to that of P. tenuis-infected animals (p>0.05; FIG. 2). Theserum sample from the Fascioloides-infected elk showed a much reducedcross-reactivity with P. tenuis L3 antigens.

[0126] 1.B.2. Anti-P. tenuis Antibodies Specifically Recognized a 37 kDaProtein

[0127] The presence of a unique P. tenuis antigen was investigated bycomparing the profile of somatic antigens of P. tenuis L3 recognized byserum antibodies from a P. tenuis-infected WTD to those recognized bythe serum antibodies from a caribou infected with E. rangiferi, and asecond caribou infected with E. rangiferi and P. andersoni concurrently.All three serum samples recognized distinct P. tenuis antigens ofapproximate mol. wt. 105, 45, 32 and 19 kDa, however only the WTD serumuniquely reacted with a P. tenuis L3 antigen of approximate mol. wt. 37kDa (FIG. 3).

[0128] Previous efforts to develop a reliable serological test for P.tenuis have been plagued by low sensitivity and poor specificity. Dew etal. (1992) used the ELISA technique to demonstrate anti-P. tenuisantibodies in the serum and cerebrospinal fluid (CSF) of goats, and inthe CSF of deer exposed to 50 P. tenuis L3 but failed to detectantibodies in the serum of infected WTD. This poor sensitivity could beattributed to the use of unfractionated antigens from adult worms.Unfractionated L3 antigen preparation appears to be more sensitive thanunfractionated adult worm preparations. Furthermore, the poorsensitivity as demonstrated by high OD backgrounds observed in thatstudy may have resulted from the use of high serum concentrations (1 in8 dilutions). The use of such dilutions may have been unavoidable giventhat caprine conjugates rather than cervid conjugates were used in theELISA. Many helminths share identical or closely related antigensresulting in cross-reactions or nonspecific reactions (Almond andParkhouse, 1985) thereby limiting the usefulness of a serological test.Neumann et al. (1994) showed that anti-P. tenuis antibodies maycross-react with even phylogenetically distant parasites likeTrichinella spiralis. The challenge of test specificity assumes greaterimportance when the parasite of interest is phylogenetically moreclosely related to other parasites present in the same geographical areaand has similar morphological characteristics and host preferences.Thus, it is important to differentiate cervids infected with P. tenuisfrom those infected with other related nematodes such as P. andersoniand E. rangiferi which are also present in North America and infectsimilar hosts. Indeed, when serum collected from caribou infected withP. andersoni and E. rangiferi were tested, they were found tocross-react strongly with P. tenuis somatic L3 antigens proving that aserological test for P. tenuis based on unfractionated, somatic L3antigens will suffer from problems of nonspecificity. As describedherein, the use of the 37 kDa fraction as the sole antigen provides amore specific test since serum samples from caribou infected with P.andersoni or E. rangiferi did not recognize the antigen by the Westernblot technique. Failure to detect the 37 kDa antigen may be due to theabsence of corresponding antibodies in the caribou sera rather than atechnical limitation with the use of anti-deer conjugate since deer andcaribou both belong to the sub-family Odocoilinae and the conjugatereacted similarly with deer IgG and caribou IgG in both ELISA andWestern blot.

[0129] 1.B.3. Adult P. tenuis Somatic Antigen Preparation ContainsUnique 37 kDa Protein

[0130] The presence of the unique 37 kDa antigen in the adult stage ofP. tenuis recognizable by serum from P. tenuis-infected WTD was assessedby immunoblotting. Infected WTD serum recognized a total of 6 adult P.tenuis antigens of which 3 antigens of approximate molecular weights 75,37 and 20 kDa were specifically and uniquely recognized (FIG. 4). About9% reactivity in the WTD serum was directed against the 37 kDa antigenas assessed by densitometric analysis. Two antigens of high molecularweights, 158 and 105 kDa, reacted nonspecifically with uninfected andinfected WTD serum samples. A 45 kDa protein present in P. tenuis, E.cervi and E. rangiferi was recognized by serum collected from aninfected deer but not by serum collected before infection.

[0131] Bienek et al. (1998) recently demonstrated extensivecross-reactivity of anti-P. tenuis antibodies with Dictyocaulusantigens. Serum collected from a WTD harboring Dictyocaulus prior toexperimental P. tenuis infection, reacted strongly with the 120 and 180kDa P. tenuis antigens but not with the 37 kDa antigen. Similarly,Neumann et al. (1994) showed that serum from a P. tenuis-infected elkwhich had a prior, natural D. viviparus infection reacted with a 36 kDaprotein present in P. tenuis but not in D. viviparus. An antigen of thesame molecular weight (37 kDa) as the unique L3 antigen was detected inthe adult parasite.

[0132] The concentrations of 37 kDa antigens present in the L3 and adultstages are not precisely known but appear to be low. When analyzed bySDS-PAGE, P. tenuis L3 and adult contain 17 and 18 protein bands,respectively. The mean concentration of proteins in P. tenuis has beenestimated at 0.11 μg per L3 and 140 μg per adult. Based on densitometricanalyses of SDS-PAGE gels, the 37 kDa antigens constitute 5.8% ofsomatic L3 antigens and 5.5% of somatic adult antigens. Theseproportions represent estimates of the maximum concentrations of theantigens in each worm stage, but actual concentrations may be lower,since a protein identified by immuno-staining may not be the onlyprotein constituent of a band of the corresponding molecular weight inan SDS-PAGE gel.

Example 2 Evaluation of Excretory-Secretory Products and Somatic WormAntigens for Diagnosis of P. Tenuis Infections

[0133] Serological diagnosis of infection based on the demonstration ofantibodies present in an infected animal relies on the availability ofgood quality antigen. The sensitivity of a serological test, i.e., howwell it detects an infected animal, and the specificity, i.e., how wellit correctly identifies animals free of the infection is largelydependent on the quality of the antigen. (Welch et al. (1991)). Theideal choice of an antigen for serological diagnosis of infectiousdiseases is one against which all infected animals produce an early andpersistent antibody response. Uninfected animals and those infected withother agents should fail to produce antibodies against the antigen.Furthermore, in nematode infections where the parasite often undergoes amultistage development in the host, the antigen should react withantibodies produced by the host in response to the different stages ofthe parasite. In order to develop a sensitive and specific serologicaltest for WTD infected with P. tenuis, three antigen preparations from P.tenuis were prepared and evaluated, namely the excretory-secretoryproducts of L3 (ES-L3) and larval and adult somatic antigens (sL3 andsA).

[0134] 2.A. Materials and Methods

[0135] 2.A.1. Parasites

[0136] Third-stage larvae (L3) of P. tenuis, obtained from pre-exposedsnails (Triodopsis multilineata) according to described methods wereused to infect white-tailed deer. (Lankester (1996)). For antigenpreparation, the L3 were either cultured to produce excretory-secretoryproducts (ES-L3) in vitro or used to prepare somatic antigens (sL3).Adult P. tenuis were obtained from the meninges of experimentallyinfected white-tailed deer at postmortem and used for preparing somaticantigens (sA). (Slomke et al. (1995)).

[0137] 2.A.2. Preparation of Parasite Antigens

[0138] L3 or adult P. tenuis were cleaned and washed in PBS, resuspendin 1 ml of PBS and kept on ice. Parasites were sonicated with aBraunsonic sonicator (Braunsonic Melungen, Allentown, Pa.) at 300 W for1 min at a time for a total of 5 min after which the parasite suspensionwas transferred into a microfuge tube and spun at 12,000 rpm for 5 min.The supernatant was kept at −20° C. Protein concentrations weredetermined using the BCA kit (Pierce, Rockford, Ill.).

[0139] ES-L3 antigens were produced in a serum-free environmentaccording to published procedures of Call et al. (1995) with somemodifications. Briefly, L3 parasites were suspended in RPMI-1640 (RPMI),transferred into a sterile dialysis bag which was knotted at the endsand placed in a petri dish containing RPMI supplemented with 10% fetalcalf serum (FCS). Parasites were cultured at 37° C. and 5% CO₂ incubator(VWR, Plainfield, N.J.) and every 24 hr thereafter, the contents of thedialysis bag were carefully aspirated, spun at 12,000 g for 5 min, andthe supernatant kept as ES antigens. To reduce the extent ofcontamination of ES-L3 by somatic antigen from degraded parasites,cultures were monitored to assess the mortality of L3. Mortality wasfound to be less than 5% for the first 7 days of uninterrupted culture.Dead and dying parasites were removed every 24 h. In addition, opticaldensity of harvested ES-L3 was measured at wavelength 260 nm to assessnucleic acid contamination and, by implication, the extent ofcontamination by degraded parasites. Nucleic acid contamination wasfound to be negligible.

[0140] 2.A.3. Infection of White-tailed Deer with P. tenuis and BloodCollection

[0141] The six WTD used in experiments were acquired as week-oldorphaned fawns in Saskatchewan where P. tenuis does not occur and raisedin captivity (Anderson et al. (1981)). They were bottle-fed and held inan open paddock until being transported to Thunder Bay, Ontario, wherethey were held on clean concrete and experimentally infected with P.tenuis L3. All animals were 9.5 months old at the start of theexperiment. Two deer were each given 6 larvae (#1 and 2), 2 were given20 (#3 and 4), and 2 were given between 100 and 150 (#5 and 6). Bloodsamples were collected from each of the WTD 1 wk before infection and atdifferent times after infection until the end of the experiment at 147days post-infection (dpi). All 6 WTD passed small numbers ofDictyocaulus larvae in their feces during the experiment but no adultspecimens could be recovered at necropsy.

[0142] 2.A.4. ELISA

[0143] Antigens (ES-L3, sL3 or sA) were diluted in PBS and adsorbed towells of a microtitre plate (Dynatech Laboratories, Chantilly, Va.) at 1μg/well and incubated at 37° C. overnight, following which wells werewashed with PBS containing 0.5% Tween 20 (PBST). Serial dilutions of WTDserum in PBST were applied to the microtitre plate wells and incubatedat 37° C. for 3 h. At the end of the incubation, plates were washed andrabbit anti-white-tailed deer IgG (Kirkegard and Perry Laboratories,Gaithersburg, Md.) diluted in PBST (1:2,000) applied to each well. Aftera 2 hour incubation at 37° C., wells of the plate were washed with PBST,phosphatase substrate (Kirkegard and Perry Laboratories, Gaithersburg,Md.) applied and allowed to react for 1 h. Development was stopped with5% EDTA and the optical density (OD) read at 405 nm with aspectrophotometer (Labsystems, Finland). For this analysis, serumsamples were divided into two batches so that each parasite dose wastested in each batch i.e., deer #1, 3 and 5 in the first batch and deer#2, 4 and 6 in the second batch. Serum samples from each batch were alltested at the same time, against ES, sL3 or sA antigens. Each sample wasanalyzed at least thrice and the results were found to be highlyreproducible. In addition, to ensure proper comparison in the presenceof possible inter-plate variation, runs were designed such that in onecase, the same microtitre plate was coated with the three differentantigens and reacted against serum samples from animals infected withthe different doses to see whether the trend observed was consistent. Inanother run, each antigen was used to coat a single plate and all sixserum samples were tested at the same time. Inter-plate and day-to-ayvariations were found to be less than 10%, therefore, results from onerepresentative batch run is presented. A post-infection serum sample wasscored positive if the ELISA OD value is equal to, or greater than,twice the OD value of the corresponding pre-infection serum sample.Statistical significance was analyzed at the 99% confidence limits.

[0144] 2.A.5. SDS-PAGE and Immunoblotting

[0145] Parasite antigens (crude L3 stage or adult worn antigens) wereseparated on a 10% SDS-PAGE at 160 V for 45 min, under reducingconditions. The separated proteins in the gel were photographedfollowing staining with BioRad Silver stain plus kit (BioRad, Hercules,Calif.) or alternatively, the proteins were transferred ontonitrocellulose membrane (BioRad, Hercules, Calif.) for immuno-staining.Membranes with proteins were blocked with PBS containing 5% milk (i.e.,PBS-milk) for 30 mins and washed with PBS containing 0.1% Tween 20(PBS-T). WTD serum samples (preinfection and 141 dpi) diluted 1 in 25 inPBST containing 2.5% milk (i.e., PBST-milk) were applied to membranestrips, and incubated overnight. Subsequently, the membrane was washedwith PBST and alkaline phosphatase-labeled rabbit anti-deer IgG diluted1 in 500 in PBST-milk added. After 4 hours of incubation, the membranewas washed three times in PBST and then a final wash in PBS. Colordevelopment ^(reagent)(BCIP/NBT phosphatase substrate) was added to themembrane and incubated in the dark. Color development was stopped after30 min by the addition of tap water to the membrane.

[0146] 2.B. Results

[0147] 2.B.1. Comparative Sensitivities of Indirect ELISAs UtilizingES-L3, sL3 or sA Antigens in the Diagnosis of P. tenuis Infections inWTD.

[0148] The ES-L3 antigen had the highest sensitivity as demonstrated bythe strongest intensity of reaction, earliest detection of antibodiesand lowest background. ES-L3 consistently detected anti-P. tenuisantibodies in all infected animals starting from the first month ofinfection until the end of the experiment (FIG. 5). The ELISA OD valuesof pre-infection sera (background OD) were consistently less than 0.14(mean=0.09). Antibodies against ES-L3 increased quickly followinginfection and attained a peak within the first month of infection (4 outof 6 animals) or soon after and often remained stable at this level (5out of 6 animals). Peak ELISA OD values of sera from the infected deerwere between 0.74 to 1.28 (mean=1.00).

[0149] The sensitivity of ES-L3 antigen in detecting antibodies to P.tenuis may be a consequence of the unique immunological reaction ofhosts to parasitic infections. Unlike viruses and bacteria that multiplyand undergo tremendous turnover in an infected host, nematodes do notdivide and while some infecting parasites may die, many that survive tothe adult stages will remain intact for a long period and may not beexposed to the immune system. P. tenuis may live for many years (Slomkeet al. (1995)), Elaphostrongylus cervi for 6 years (Watson (1984)) andP. odocoilei for 9.4 years (Samuel W. M, personal communications). Thus,the main immunological recognition of a parasite by the host may be viathe ES-L3 products produced by the parasite and consequently, thepredominant immune response directed against the ES products.

[0150] Similar to ES-L3 antigen, the sL3 antigen consistently reactedwith anti-P. tenuis antibodies in all infected animals throughout thecourse of infection. Anti-sL3 antibodies were also detectable inresponse to infection from the first month (FIG. 5). The background ODlevels for sL3 ranged from 0.15 to 0.35 (mean=0.21). Peak ELISA ODvalues ranged from 0.47 to 0.78 (mean=0.66). Anti-sL3 antibodiesdecreased terminally in 4 of 6 infected animals, but remained detectablein all animals until the end of the experiment. The presence of anti-sL3antibodies late in the infection and at a time when L3 stages would havematured into adult forms may be explained by two different events.First, it appears that the anti-sL3 antibodies are long-lived assupported by the finding that serum collected at the termination of theexperiment, and used in immunoblotting, recognized an antigen (M. Wt.=40kDa.) present in the somatic antigen preparation of larvae but not inthe adult worm preparation. Second, cross-reacting antigens present inadult antigens may boost the initial anti-sL3 response as suggested bythe possible recognition of 37 kla antigens in larval and adult antigenpreparations.

[0151] In contrast to ES-L3 and sL3, the sA antigen detected anti-P.tenuis antibodies in 4 infected animals but not in two animals (#1 and3) infected with 6 and 20 parasites (FIG. 5). In one animal (#3), thebackground OD value (=0.55) was higher than the OD value of any of thepost-infection sera collected on 4 different occasions between 28 and141 dpi (OD range=0.35 to 0.4). The background OD for sA antigen rangedfrom 0.16 to 0.55 (mean=0.30). The peak OD values ranged from 0.37 to0.75 (mean=0.5). Non-specific background reactions accounted for 9% ofthe anti-ES, 32% of anti-sL3, and 60% of anti-sA reactivities.

[0152] Thus, the sA antigen was the least sensitive antigen preparation.It detected anti-P. tenuis antibodies within one month of infection intwo animals (#2 and 5), after the first month in two other animals (#4and 6) and at no time throughout the course of infection in the tworemaining animals (#1 and 3). Even among the positive animals, theintensity of reaction as assessed by OD readings, was relatively low.These observations confirm previous suggestions (Bienek et al. (1998);Neumann et al. (1994)) that adult somatic antigens may not be veryuseful in the diagnosis of P. tenuis. The effectiveness of ES productsfrom the adult worm could not be assessed because live adult worms werenot available at the time of the study.

[0153] ES-L3 provided the greatest discrimination between the ELISA ODvalues of pre- and post-infection sera. ES-L3 detected antibodies to P.tenuis in all infected animals within the first month of infection andat all sampling times thereafter. The intensity of the earliestreactions was higher with ES-L3 than sL3. ES-L3 appears to be superiorto either sL3 or sA in the diagnosis of P. tenuis infection sinceanti-ES-L3 antibodies were induced very quickly in infected animals andwere generally maintained at stable high levels during the course of theexperiment.

[0154] In order to test which antigen could most sensitively detect theearly presence of WTD anti-P. tenuis antibodies, serum samples collectedprior to infection, and at 14 or 28 dpi were tested against ES, sL3 orsA antigen by indirect ELISA. ES-L3 and sL3 detected anti-P. tenuisantibodies in serum at 14 dpi (FIG. 2). Furthermore, ES-L3 and sL3showed a fourfold increase in the intensity of reaction betweenpre-infection and the serum sample collected 14 dpi. The sA antigenfailed to detect antibodies in infected animals at day 14 dpi. Theintensity of the early reaction (14 and 28 dpi) was highest for ES-L3and lowest for sA.

[0155] Anti-ES-L3 antibodies were detectable by ELISA and Immunoblottingat the terminal stages of the experiment. Anti-ES-L3 antibodies, similarto anti-sL3 antibodies, persisted in all infected animals long after theL3 stages would have moulted into advanced stages, and by implication,the cessation of ES-L3 production. The persistence of antibodies couldbe explained by mechanisms similar to that of anti-sL3: longevity ofantibodies and identical antigens produced by more advanced parasitestages.

[0156] The suitability of the sL3 in serological diagnosis of P. tenuismay be attributed to the common antigens present in the infective L3stage and their ES products. Interestingly, infected WTD serumrecognized at least three antigens present in ES-L3 but not in sL3,suggesting that once these antigens are produced by L3, they arecompletely excreted. This observation discounts the possibility ofsignificant cross-contamination of the ES-L3 antigen preparation by sL3antigen. An antigen found to be abundant in the ES products whileapparently undetectable in the somatic antigen preparation may be moreappropriately described as an excretory antigen while an antigen sharedbetween the ES products and the somatic antigen preparation may bedescribed as a secretory antigen.

[0157] 2.B.2. Cross-reactivity of ES-L3, sL3 and sA with Sera fromCervids Infected with Other Parasites

[0158] ES-L3, sL3 and sA showed significant cross-reactivity with serafrom red deer infected with E. cervi, and from caribou infected with E.rangiferi only, or concurrently with P. andersoni (FIG. 7). The ES-L3showed the strongest cross-reactivity among the three antigenpreparations. Serum from E. rangiferi-infected caribou showed the least,but nevertheless significant, cross-reactivity.

[0159] All three antigen preparations show low specificity asdemonstrated by high cross-reactivity with sera from cervids infectedwith parasites closely related to P. tenuis, namely P. andersoni, E.rangiferi and E. cervi. The observed cross-reactions are indications ofthe similarities between some antigens of P. tenuis and those found inthe other parasites. The report that P. tenuis share as many as 5antigens with Trichinella spiralis (Family: Trichuridae) and resultingin cross-reactivity between these two parasites (Neumann et al., 1994)further illustrates the challenges of developing a specific serologicaltest not only for P. tenuis but for parasites, using unfractionatedantigens. Thus, in spite of the sensitivity of ES-L3 or sL3 antigens, itis evident that unfractionated P. tenuis antigen may not be useful forfield identification of P. tenuis-infected cervids. It is thereforeimperative to identify individual antigen(s) unique to the parasite. Asdescribed in Example I above, identification of a unique 37 kDa proteinof P. tenuis that reacts with P. tenuis-infected WTD serum but not serafrom cervids infected with E. rangiferi, P. andersoni or E. cervi isdescribed. A protein of about 37 kDa size appears to be present in allthree antigen preparations (FIG. 9). It is not clear why the 37 kDa inthe adult antigen failed to produce a positive result in animals #1 and3; possible explanations include low levels of antibodies in infectedanimals or high non-specific reactivity of pre-infection sera to otherantigens. A more probable reason is the complexity of the adult antigenpreparation, as shown by a higher number of individual antigens than forthe larval antigens, which may be a confounding factor for specificantigen-antibody interactions. The nature of the antigen appears todetermine the extent of false positive serological results (see, e.g,Gamble et al. (1993).

[0160] In sum, previously untested excretory-secretory products from P.tenuis showed the best sensitivity and may be used, as the larvalsomatic antigens, for monitoring experimental P. tenuis infections.

[0161] 2.B.3. SDS-PAGE Analysis of ES-L3. sL3 or sA

[0162] Electrophoretic separation of the antigens done to analyze thecomplexity and the size compositions of individual proteins present ineach of the antigen preparations showed that the ES-L3 antigenpreparation had the least number of protein bands and sA the most. Sixprotein bands were detected in the ES-L3 with a m. wt. range of 21-72kDa. The sL3 had 15 protein bands with a m. wt. range of 14-97 kDa whilesA had 26 protein bands and a m. wt. range of 14-116 kDa. (FIG. 8).

[0163] 2.B.4. Western Blot Analysis of P. tenuis-infected Serum AgainstES-L3, sL3 or sA.

[0164] Infected deer serum recognized antigens of different sizes ineach antigen preparation, however, a 37 kd appeared to be the onlyprotein consistently recognized by P. tenuis-infected deer serum in allthree antigen preparations. As in the ELISA, the highest backgroundreaction observed in Western blot was with sA (FIG. 9).

[0165] Thus, antigens of the third-stage larva may be more relevant thanthose from adults in the serodiagnosis of P. tenuis and furthermore,excreted and secretory antigens are more sensitive than the somaticantigens of the third-stage larvae.

Example 3 Construction, Screening of cDNA Library and Identification ofParasite Genes

[0166] 3.A. Materials and Methods

[0167] 3.A.1. Isolation of P. tenuis RNA

[0168] Adult P. tenuis worms (males and females) were washed three timesin phosphate buffered saline (PBS, pH 7.4) and disrupted by passingthrough the barrel of a syringe and applied to a shredder (Qiagen, SantaClarita, Calif.) according to the manufacturers instructions. Total RNAwas isolated from disrupted P. tenuis worms using the RNeasy mini kit(Qiagen, Santa Clarita, Calif.).

[0169] 3.A.2. Antiserum

[0170] Mouse anti-P. tenuis antiserum was produced by immunization often mice with sonicates of P. tenuis adult worm. White-tailed deer (WTD)anti-P. tenuis antiserum was obtained from a white-tailed deer infectedwith 100 P. tenuis third-stage larvae. Serum obtained from the WTDbefore infection was used as control (normal) serum. Elkanti-Dictyocaulus antiserum was obtained from an elk naturally infectedwith Dictyocaulus viviparus. Rabbit anti-E. cervi antibodies wasproduced by immunizing a rabbit with sonicated adult E. cervi.

[0171] 3.A.3. Construction of cDNA Library

[0172]P. tenuis cDNA was synthesized using a cDNA kit, and a libraryconstructed with the UNIZAP vector (Stratagene, La Jolla, Calif.),according to the manufacturer's instructions. Using total RNA asstarting material, P. tenuis messenger RNA was reverse transcribed tofirst strand cDNA by use of XhoI linker-poly(dT) primer, Moloney murineleukemia virus (MMLV) reverse transcriptase, and a ribonucleaseinhibitor at 37° C. for 1 hour. Second strand cDNA was synthesized byEscherichia coli DNA polymerase following the generation of nicks in RNAof the first strand cDNA-RNA duplex by RNase H. cDNA synthesis wasmonitored by the addition of radioactive nucleotides in controlreactions containing aliquots of the first or second-strand reactionmix. The synthesized, double-stranded cDNA was blunt ended with clonedpfu DNA polymerase at 72° C. for 30 minutes, ligated to EcoRI adapterswith T4 DNA ligase at 4° C. for 48 hours, and digested with Xho I at 37°C. for 1.5 hours, resulting in cDNA with an XhoI cohesive 5′ end, and anEcoRI cohesive 3′ end.

[0173] The cDNA was separated according to size by chromatography onSepharose CL-2B, and larger sized cDNA fragments were ligatedunidirectionally into a UNIZAP vector that was predigested with Xho Iand EcoRI, and packaged into Gigapack III Gold packaging extract(Strategene). The titer of, and the percent recombinants in, the primarylibrary were determined by infecting E. coli XLI-Blue MFR with analiquot of the cDNA-UNIZAP library (phage), and mixing with 0.7%agarose-LB (Top agar), Isopropyl-1-thio-β-D-galactopyranoside (IPTG) and5-Bromo4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) before platingon a NZY agar plate. The primary library was amplified once in E. coliXLI-Blue MRF and stored at −70° C.

[0174] 3.A.4. Immunoscreening of cDNA Library

[0175] Primary screening of the amplified cDNA-UNIZAP library was donewith hyperimmune mouse anti-P. tenuis (adult worm) antiserum. First, thecDNA-UNIZAP library was plated at an average density of 4,500 PFU per150-mm agar plate, and incubated at 37° C. for 5 h or until plaques arebarely visible. Nitrocellulose, pre-soaked in 10 mM IPTG solution wasapplied to the plate and incubated for a further 4 h. The membrane wasremoved, rinsed in PBS, pH 7.4, and placed in a blocking solution of PBScontaining 20% fetal calf serum (FCS) for 2 h at room temperature.Following the blocking step, nitrocellulose was rinsed in PBS andincubated at room temperature, overnight, in mouse antiserum solution(1:1000) in PBS containing 0.1% Tween 20 (PBST). Nitrocellulose waswashed in PBST, thrice and placed in a solution of peroxidase-labeledrabbit anti-mouse IgG, diluted 1:1000 in PBST and allowed to react for 4h at room temperature. At the end of the incubation, nitrocellulose waswashed three times in PBST and developed with phosphatase substrate(5-Bromo4-chloro-3-indolyl-phosphate-Nitro Blue Tetrazolium, Kirkegaard& Perry Laboratories, Gaithersburg, Md.).

[0176] 3.A.5. Identification of Positive Clones

[0177] Positive reacting plaques were picked by orientating markednitrocellulose against the agar plate and picking the plaques as an agarplug with the end of a sterile pasteur pipette. Phages in the plug wereeluted into SM buffer (50 mM Tris, pH 7.5 containing 0.1M NaCl, 0.01%gelatin) by overnight incubation on a shaker. Eluted phages were platedand re-screened as above until a population of uniformly reactingpositive clones were obtained. Each positive clone was amplified andstored at −70° C. Further screening of clones was done with serum fromP. tenuis-infected white-tailed deer. The specificity of antigenexpressed by the putative clones was assessed by using sera specific forother parasites (red deer anti-Elaphostrongylus cervi, elkanti-Dictyocaulus viviparus) on the nitrocellulose.

[0178] 3.A.6. PCR Amplification of Positive Clones

[0179] Confirmation of the presence of, and the determination of thesize of insert in each clone, were done by PCR of the multiple cloningsite of the UNIZAP vector between the T3 and T7 sequences. The PCRreaction consists of lysis of cDNA-UNIZAP clone (10⁷ phages) at 95° C.for 5 min followed by annealing of the vector DNA with T3 and T7 primers(0.5 μM per primer) at 55° C. for 5 min. Hot-start PCR was performedusing Taq polymerase (AmpliTaq, Perkin Elmer) activated at 95° C. for 10min and reaction mixture was subjected to each of 40 cycles consistingof a primer extension (72° C., 90 s), denaturation (94° C., 30 s),annealing (55° C., 75 s) and a final primer extension step (72° C., 10min).

[0180] 3.A.7. Sizes of Cloned P. tenuis Genes

[0181] The size of the cDNA insert present in each UNIZAP-cDNA clone wasdetermined by PCR amplification of the insert using T3 and T7 primerswhich bind regions of the UNIZAP vector at either ends of the insert.UNIZAP-cDNA clone 128, 175, 212, and 323 were previously demonstrated toproduce proteins that react with anti-P. tenuis antibodies. Clone 212also reacted with anti-E. cervi antibodies. Clones of UNIZAP vector only(i.e., without insert=1′, 2′ and 3′) were included as controls. Aliquots(10 μl) from the PCR reaction tubes were analyzed on 0.7% agarose gelsand visualized by ethidium bromide staining.

[0182] 3.B. Results

[0183] 3.B.1. P. tenuis RNA

[0184] Between about 21 and about 25 μg of total RNA was extracted froma total of 6 adult P. tenuis Worms.

[0185] 3.B.2. cDNA Synthesis

[0186] The synthesized cDNA ranged from 0.1-4 kb in length. FIG. 10shows the messenger RNA component of P. tenuis total RNA was transcribedinto first (Lane 1) and second (Lane 2) strand cDNA using MMLV reversetranscriptase. cDNA synthesis was monitored by the incorporation ofradioactive nucleotides, agarose analysis and visualization byradiographic analysis. In FIG. 10, M=λ-HindIII molecular weight marker.

[0187] 3.B.3. Size of P. tenuis cDNA Library

[0188] Following the ligation of P. tenuis cDNA with the UNIZAP vectorto create the P. tenuis cDNA-UNIZAP primary library, the titre of thelibrary was estimated at 1.13×10⁵ clones.

[0189] 3.B.4. Immunoreactive P. tenuis Clones

[0190] Thirteen clones were found to react with mouse anti-P. tenuisantiserum. FIG. 11 shows identification of P. tenuis cDNA clones fromprimary screening with mouse anti-P. tenuis antiserum and re-screenedwith other sera/antisera (2. WTD anti-P. tenuis, 3. WTD normal serum, 4.Elk anti-Dictyocaulus) to assess cross-reactivity or uniqueness ofclones. Four of the clones, 191, 210, 212 and 222 reacted with mouseanti-P. tenuis and rabbit anti-E. cervi antibodies.

[0191]FIG. 12 shows screening of amplified P. tenuis cDNA-UNIZAP libraryon nitrocellulose with mouse anti-P. tenuis antiserum to identify P.tenuis antigen-producing clones. E. coli XLI was infected with 6×10³ P.tenuis cDNA-UNIZAP phages of the amplified library, and grown on an NZYplate. Plaques produced by the lysis of bacteria were transferred tonitrocellulose and the presence of putative P. tenuis antigensidentified with the antiserum. One plaque (arrow in FIG. 12) producedantigen that reacted with anti-P. tenuis antiserum.

[0192] Size of P. tenuis Genes

[0193] The size of the P. tenuis genes that produce antigens thatreacted with anti-P. tenuis antiserum as well as anti-E. cerviantiserum, as determined by PCR is as follows: Clone Size of gene (bp)191 not determined 210 575 212 875 222 1,375

[0194] In addition, FIG. 13 shows the size of cDNA inserts present inUNIZAP-clones 128, 175, 212 and 323 as determined by PCR amplificationusing T3 and T7 primers. The lane labeled “V1” is the molecular weightmarker. Lanes labeled 1′, 2′ and 3′ are UNIZAP vector only controls. Thesize of the P. tenuis gene PCR produce minus the contribution of primersand vector nucleotides. All clones produce polypeptides that react withanti-P. tenuis-antibodies. Clone 212 also reacted with anti E. cerviantibodies.

[0195] Thus, polynucleotides encoding Protostrongylidae antigens (bothcommon and P. tenuis-specific antigens) were isolated, sized andscreened for reactivity.

Example 4 Serological Diagnosis of Elaphostrongylus cervi Infection andIdentification of a Unique E. cervi Antigen

[0196] 4.A. Materials and Methods

[0197] 4.A.1. Parasites and Sera

[0198] Red deer were infected with the L3 stages of E. cervi obtainedfollowing published methods (Gajadhar and Tessaro (1995) J. ofParasitology 81:593-596). Originally, first-stage larvae of E. cerviobtained from infected red deer in New Zealand were exposed tolaboratory-reared snails (Triodopsis multilineaia) and slugs (Derocerosreticulatum) and the third stage larvae obtained from the gastropodswere used to infect red deer. Six weeks after exposure, slugs or snailswere placed in a 300 μl sieve (Canadian Standard Sieve Series, WS Tyler,St. Catharines, Ontario, Canada) and immersed in a beaker of 250 ml of0.7% pepsin in water containing 0.8% HCl at 37° C. for 1 h. At the endof the incubation, the L3 present in the pepsin-HCl were allowed tosediment, washed 3 times in water, picked individually under adissecting microscope (Leica MS5, Heerbrugg, Switzerland) and counted.

[0199] 4.A.2. Animals and Infection

[0200] Orphaned red deer fawns kept in isolation were bottle-raised andweaned onto a pelleted ration. Animals were infected per os with L3stages of E. cervi. Feces were collected daily from the fawns andanalyzed by a modification of the Baermann's procedure for theexamination of E. cervi L1 (Gajadhar et al. (1994) Canadian VeterinaryJournal 35:433-437) to detect the onset of passing of larval stages,i.e., patency. Blood samples were collected from the fawn before, and atregular intervals after infection. Animals were restricted in their pensaccording to the guidelines of the Canadian Council on Animal Care. Thecare of the animals and the handling of fomites, equipment and deerfeces were done according to Institutional guidelines to prevent thecontamination of the environment with E. cervi.

[0201] 4.A.3. E. cervi Antigens

[0202] ES products: 1000 L3 obtained from slugs or snails and pickedclean were cultured in RPMI-1640 containing gentamycin (100 μg/ml) andfungizone (10 μg/ml) at 37° C. and with 5% CO₂. At 4 day intervals,culture supernatant containing parasite ES product was removed from theculture and the following protease inhibitors added: EDTA (2 mM),leupeptin (2 μg/ml), pepstatin (1 μg/ml), phenylmethanesulphonylfluoride, PMSF (100 μg/ml), tosyl-L-lysine chloromethyl ketone, TLCK (50μg/ml), and tosyl-amido-1-phenylethyl chloromethyl ketone, TPCK, (100μg/ml). The culture supernatant was spun at 12,000 g for 10 min tosediment insoluble parasite material. The culture supernatant containingES products was then concentrated using Centricon-3 and Microcon-3 (Mwtcut off=3 kDa; Amicon Inc., Beverly, Md.). When it is necessary to knowthe protein concentration of the ES products, protease inhibitors werenot added and the culture supernatant was dialyzed against PBS, pH 7.4overnight at 4° C. before concentration and protein concentration wasdetermined with the aid of the Bicinchoninic acid, BCA, kit (Pierce,Rockford, Ill.).

[0203] L3 crude antigens: L3 obtained from slugs or snails (as above)were suspended in PBS and sonicated (Braunsonic, Melsungen AG) at 300 W1 min, rested for 1 min and repeated until worms were sonicated for atotal period of 5 min. At the end of the sonication, no discernable wornfragments were present in the worm suspension which was then transferredinto a microfuge tube and spun at 12,000 g for 5 min. The supernatantwas kept at −20° C. Protein concentration was determined using the BCAkit.

[0204] 4.A.4. SDS-PAGE and Immunoblotting

[0205] Antigen (ES products or L3 crude antigens) were separated on a10% SDS-PAGE at 160 V for 45 min, under reducing conditions. At the endof the run, gels were stained with Coomassie stain or Silver stain kit(BioRad, Hercules, Calif.) to visualize the protein bands oralternatively, the proteins in unstained gels were transferred ontonitrocellulose membrane (BioRad, Hercules, Calif.) for immunostaining.Membranes bearing proteins were blocked with PBS containing 5% milk(i.e., PBS-milk). Serum samples (deer or control) diluted 1 in 25 in PBScontaining 0.1% Tween 20 (PBS-T) and 2.5% milk (i.e., PBST-milk) wereapplied to membrane strips and incubated overnight. After incubation,the membrane was washed with PBST and the appropriate conjugate (e.g.,alkaline phosphatase-labeled rabbit and anti-deer IgG) diluted 1 in 500in PBST-milk added. After 4 hours of incubation, membrane was washedthree times in PBST and then a final wash in PBS. Color developmentreagent (BCIP/NBT phosphatase substrate, Kirkegaard and PerryLaboratories, Gaithersburg, Md.) was added to the membrane and incubatedin the dark. Color development was stopped after 30 min by the additionof tap water to the membrane.

[0206] 4.B. Results

[0207] 4.B.1. ELISA Anti-E. cervi Antibodies

[0208] Serum antibodies to E. cervi L3 were detectable in all threeinfected animals at 23 days post-infection as assessed by indirectELISA. Antibodies levels increased steadily up to 42-71 dpi and declinedthereafter in all animals. Antibodies dropped to undetectable levels by176 dpi in the animals given 6 parasites. In the other animals,antibodies persisted until the termination of experiment at 176 or 537dpi.

[0209] 4.B.2. SDS-PAGE Analysis of the Excretory/Secretory and L3Somatic Antigens of Elaphostrongylus cervi

[0210] Electrophoretic analysis of the constituents of E. cervi ESproducts revealed the presence of 9 protein bands with a broad range ofmolecular weights (<14.4 to over 200 kDa). In comparison, the L3consists of as many as 16 protein bands also with a broad range ofmolecular weights (19.1-97.4 kDa). The ES products contain 2 bands thatare significantly greater in sizes than the heaviest L3 antigen band.

[0211] 4.B.3. Anti-E. cervi Somatic L3 Antibodies

[0212] Infected deer serum reacted with eight protein bands of E. cerviL3 following separation of crude L3 antigen by SDS-PAGE andimmunoblotting. The eight protein bands had a molecular weight range of<19 kDa to 160 kDa under reducing conditions. Serum samples from theanimal infected with 100 L3 (Deer #3) showed the most distinct patternof reactivity. Two parasite proteins band of molecular weights 19 and 35kDa reacted slightly with pre-infection serum but showed a strongerreaction with post-infection sera collected on day 23 and 211 afterexposure to L3. A 45 kDa protein reacted strongly with post-infectionsera samples while a 47 kDa protein reacted less significantly andsimilarly, only with post-infection serum sample. Three protein bands(52-78 kDa) did not react with pre-infection sera but reactedsignificantly with day 211 post-infection sera but less strongly withpost-infection sera collected earlier.

[0213] Sera samples collected from deer #2 post-infection detected mostof the proteins but to a lesser degree for the larger protein bands of47 kDa and 52-78 kDa. The 19 and 35 kDa bands were detectable bypost-infection sera as in deer #3, but were negative for thepre-infection sera.

[0214] Deer #1 sera did not react with the 47 kDa protein at any time.The strong staining 35 kDa protein strongly reacted with pre-infectionserum sample.

[0215] The 35 kDa behaves as an immunodominant antigen and shows verystrong cross-reactivity with pre-infection serum sample from one animal(deer #1).

[0216] 4.B.4. Anti-E. cervi ES Antibodies

[0217] Infected deer (#3) sera reacted with 5 distinct ES antigen bands.Two of these antigen bands reacted non-specifically with pre-infectionserum, but the intensity of the reaction increased with infected serasamples. The reaction was strongest in sera collected 23 dayspost-infection. The bands recognized had estimated molecular weights of37, 42, 45, 63 and 128 kDa. In addition, there was a strong band at therunning end of the gel (front dye) which may correspond to a proteinsize of 19 kDa or less or may represent smaller fragments from any ofthe bigger proteins. Sera from all 3 infected animals show similarpattern of reactivity with ES antigens. FIG. 14 shows the identificationof a unique 52 kDa E. cervi-specific antigen.

[0218] Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

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What is claimed is:
 1. An isolated immunogenic Protostrongylidae antigenselected from the group consisting of a P. tenuis-specific 20 kDaantigen, a P. tenuis-specific 37 kDa antigen, an E. cervi-specific 37kDa antigen, a 52 kDa antigen and a P. tenuis-specific 75 kDa antigen, acommon 105 kDa antigen or a common 158 kDa antigen, as determined bySDS-PAGE gel electrophoresis and immunoblotting.
 2. An isolated antibodythat recognizes an epitope on an antigen of claim
 1. 3. A polynucleotideencoding an antigen according to claim
 1. 4. An isolated immunogenic P.tenuis-specific antigen.
 5. A P. tenuis antigen according to claim 4selected from the group consisting of a P. tenuis-specific 20 kDaantigen, a P. tenuis-specific 37 kDa antigen and a P. tenuis-specific 75kDa antigen, as determined by SDS-PAGE gel electrophoresis.
 6. A P.tenuis-specific antigen according to claim 4 wherein the P.tenuis-specific antigen is a 37 kDa antigen, as determined by SDS-PAGEgel electrophoresis.
 7. An isolated antibody that recognizes an epitopeon the antigen of claim
 4. 8. A polynucleotide encoding an antigenaccording to claim
 4. 9. A polynucleotide encoding an antigen accordingto claim
 6. 10. An isolated immunogenic E. cervi-specific antigen. 11.An E. cervi-specific antigen according to claim 10 selected from thegroup consisting of a E. cervi-specific 37 kDa antigen and a E.cervi-specific 52 kDa antigen, as determined by SDS-PAGE gelelectrophoresis.
 12. An E. cervi-specific antigen according to claim 10wherein the E. cervi-specific antigen is a 37 kDa antigen, as determinedby SDS-PAGE gel electrophoresis.
 13. An isolated antibody thatrecognizes an epitope on the antigen of claim
 10. 14. A polynucleotideencoding an antigen according to claim
 10. 15. A polynucleotide encodingan antigen according to claim
 12. 16. A method of diagnosingProtostrongylidae infection in a vertebrate subject, comprisingdetecting the presence of at least one common Protostrongylidae antigenin a biological sample obtained from the subject, wherein the presenceof the at least one common antigen is indicative of Protostrongylidaeinfection.
 17. The method of claim 16 wherein the method comprisesdetecting the presence of a common 105 kDa antigen or a common 158 kDaantigen.
 18. The method of claim 16, wherein the method comprisesdetecting the presence of more than one common antigen.
 19. The methodof claim 18, wherein the method comprises detecting the presence of acommon 105 kDa antigen and a common 158 kDa antigen.
 20. The method ofclaim 16, wherein the at least one common antigen is detected using anantibody.
 21. The method of claim 16, wherein the at least one commonantigen is detected using a nucleic acid probe.
 22. The method of claim16, wherein the at least one common antigen is detected using PCR. 23.The method of claim 16, wherein the Protostrongylidae infection iscaused by P. tenuis or E. cervi.
 24. The method of claim 20, wherein themethod comprises: (a) reacting the biological sample with one or moreisolated common Protostrongylidae antigens under conditions which allowanti-Protostrongylidae antibodies, when present in the sample, tospecifically bind with said common antigens; (b) removing unboundantibodies; (c) providing one or more moieties capable of associatingwith the bound antibodies; and (d) detecting the presence or absence ofthe one or more moieties, thereby detecting the presence or absence ofProtostrongylidae infection.
 25. The method of claim 24, wherein the oneor more moieties comprises a detectably labeled immunoglobulin antibody.26. The method of claim 24, wherein the one or more Protostrongylidaecommon antigens are a common 105 kDa antigen or a common 158 kDaantigen.
 27. The method of claim 16, wherein the biological sample is aserum sample.
 28. A method for specifically diagnosing P. tenuisinfection in a subject, the method comprising detecting the presence ofone or more P. tenuis-specific antigens in a biological sample obtainedfrom the subject.
 29. The method of claim 28, wherein the one or more P.tenuis-specific antigens are detected using an antibody.
 30. The methodof claim 28, wherein the one or more P. tenuis-specific antigens aredetected using nucleic acid hybridization.
 31. The method of claim 28,wherein the one or more P. tenuis-specific antigens are detected usingPCR.
 32. The method of claim 28, wherein the method comprises, (a)reacting the biological sample with one or more isolated P.tenuis-specific antigens under conditions which allow antibodies, whenpresent in the sample, to specifically bind with the specific antigens;(b) removing unbound antibodies; (c) providing one or more moietiescapable of associating with the bound antibodies; and (d) detecting thepresence or absence of the one or more moieties, thereby detecting thepresence or absence of P. tenuis infection.
 33. The method of claim 32,wherein the one or more moieties comprises a detectably labeledimmunoglobulin antibody.
 34. The method of claim 28, wherein the one ormore P. tenuis-specific antigens are selected from the group consistingof a P. tenuis-specific 20 kDa antigen, a P. tenuis-specific 37 kDaantigen and a P. tenuis-specific 75 kDa antigen.
 35. The method of claim28, wherein the P. tenuis specific antigen is a P. tenuis-specific 37kDa antigen.
 36. The method of claim 28, wherein the biological sampleis a serum sample.
 37. A method for specifically diagnosing E. cerviinfection in a subject, the method comprising detecting the presence ofone or more E. cervi-specific antigens in a biological sample obtainedfrom the subject.
 38. The method of claim 37, wherein the one or more E.cervi-specific antigens are detected using an antibody.
 39. The methodof claim 37, wherein the one or more E. cervi-specific antigens aredetected using nucleic acid hybridization.
 40. The method of claim 37,wherein the one or more E. cervi-specific antigens are detected usingPCR.
 41. The method of claim 37, wherein the method comprises, (a)reacting the biological sample with one or more isolated E.cervi-specific antigens under conditions which allow antibodies, whenpresent in the sample, to specifically bind with the specific antigens;(b) removing unbound antibodies; (c) providing one or more moietiescapable of associating with the bound antibodies; and (d) detecting thepresence or absence of the one or more moieties, thereby detecting thepresence or absence of E. cervi infection.
 42. The method of claim 41,wherein the one or more moieties comprises a detectably labeledimmunoglobulin antibody.
 43. The method of claim 37, wherein the one ormore E. cervi-specific antigens are 37 kDa or 52 kDa antigens.
 44. Themethod of claim 37, wherein the E. cervi-specific antigen is a 37 kDaantigen.
 45. The method of claim 37, wherein the biological sample is aserum sample.
 46. A method of detecting antibodies to parasites in abiological sample, comprising (a) reacting the biological sample with anantigen preparation selected from the group consisting of an ES-L3antigen preparation and an sL3 antigen preparation, under conditionswhich allow parasitic antibodies to bind to an antigen in the antigenpreparations and form an antigen:antibody complex; and (b) detecting thepresence or absence of said complex, thereby detecting the presence orabsence of parasitic antibodies in said biological sample.
 47. Themethod of claim 46 wherein the parasites are Protostrongylidae.
 48. Akit for use in the diagnostic method according to claim 28, comprisingin a suitable packaging: one or more common or P. tenuis- or E.cervi-specific antigens immobilized on a solid support; and a reagentsuitable for detecting, in a biological sample, the presence ofantibodies to the one or more common or P. tenuis- or E. cervi-specificantigens.
 49. A kit for use in the diagnostic method according to claim37, comprising in a suitable packaging: one or more common or P. tenuis-or E. cervi-specific antigens immobilized on a solid support; and areagent suitable for detecting, in a biological sample, the presence ofantibodies to the one or more common or P. tenuis- or E. cervi-specificantigens.
 50. An antigen according to claim 1 obtained by: (a) providinga cDNA library which expresses protostrongylidae genes; (b) screeningthe expressed genes of the cDNA library with a source ofanti-protostrongylidae antibodies to identify cDNA clones which expresscommon antigens; and (c) transforming a host cell with the cDNA cloneswhich express the common antigen, thereby producing the antigen.
 51. Anantigen according to claim 4 obtained by: (a) providing a cDNA librarywhich expresses P. tenuis genes; (b) screening the expressed genes ofthe cDNA library with a source of anti-P. tenuis antibodies to identifycDNA clones which express P. tenuis-specific antigens; and (c)transforming a host cell with the cDNA clones which express the P.tenuis-specific antigen, thereby producing the antigen.
 52. An antigenaccording to claims 10 obtained by: (a) providing a cDNA library whichexpresses E. cervi genes; (b) screening the expressed genes of the cDNAlibrary with a source of anti-E. cervi antibodies to identify cDNAclones which express E. cervi-specific antigens; and (c) transforming ahost cell with the cDNA clones which express the E. cervi-specificantigen, thereby producing the antigen.
 53. A method of isolating one ormore common Protostrongylidae antigens, comprising: (a) purifyingproteins from the excretory-secretory (ES) Protostrongylidae; and (b)determining ES purified proteins that react with anti-Protostrongylidaeantibodies, thereby isolating one or more common Protostrongylidaeantigens.
 54. A method of isolating one or more P. tenuis-specificantigens, comprising: (a) purifying proteins from theexcretory-secretory products (ES) of P. tenuis; and (b) determining ESpurified proteins that react with anti-P. tenuis antibodies, therebyisolating one or more P. tenuis-specific antigens.
 55. A method ofisolating one or more E. cervi-specific antigens, comprising: (a)purifying proteins from the excretory-secretory products (ES) of E.cervi; and (b) determining ES purified proteins that react with anti-E.cervi antibodies, thereby isolating one or more E. cervi-specificantigens.